WO2023106513A1 - Bio-patch-type cell therapy product for skin regeneration, comprising placenta-derived stem cells - Google Patents
Bio-patch-type cell therapy product for skin regeneration, comprising placenta-derived stem cells Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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Definitions
- the present invention relates to a biopatch-type cell therapy product for skin regeneration containing placenta-derived stem cells.
- Stem cells are undifferentiated cells that are capable of self-replication and can differentiate and proliferate from one cell into cells constituting various tissues. They have a unique ability to regenerate damaged body tissues and cells. there is. It is recognized and known as a next-generation technology with high medical applicability for difficult-to-treat injuries such as degenerative diseases for which there is currently no definite treatment or severe trauma, and clinical trials and research using stem cells are being conducted in the field of regenerative medicine.
- Stem cells are largely classified into embryonic stem cells and adult stem cells according to the developmental state of the tissue of origin.
- induced pluripotent stem cells using gene editing technology is there.
- Regenerative treatment research using stem cells mainly uses mesenchymal stem cells, a type of adult stem cells, in consideration of the ease of obtaining tissue raw materials, the efficiency of stem cell isolation, and the problem of tumor formation. It can be obtained from tissues such as fat and placenta.
- Placental stem cells are stem cells derived from umbilical cord blood, amnion, and amniotic fluid, and have been recently discovered and studied among other tissue-derived mesenchymal stem cells. Placental stem cells have excellent proliferative ability, have no tumor formation problem, and have the ability to differentiate into mesodermal, endodermal, and ectodermal tissues such as fat, bone, muscle, liver, and nerve. In particular, it is the only stem cell that has overcome ethical problems, and has the advantage of being non-invasively obtained at birth. shows potential as a cell therapy.
- Figure 1 is a state of transfer of equine amnion tissue to a sterile culture medium.
- Figure 2 shows the results of initial culture of the amnion-derived cells of the present invention according to one embodiment of the present invention.
- Figure 3 confirms the ability of self-replication and proliferation of the amniotic membrane-derived stem cells of the present invention according to an embodiment of the present invention. This is a graph comparing the doubling time of stem cells and skin-derived fibroblasts.
- A before differentiation induction (X100), B: after 7 days of culture (X100), C: after 7 days of culture (X200))
- FIG. 6 is a result of confirming the differentiation ability of the amniotic membrane-derived stem cells of the present invention into osteoblasts according to an embodiment of the present invention.
- A before differentiation induction (X100), B: after 7 days of culture (X100), C: after 7 days of culture (X200))
- RT-PCR reverse transcription polymerase chain reaction
- FIG 11 shows the shape of a porous film cell culture surface manufactured according to an embodiment of the present invention.
- Figure 12 confirms the cell engraftment rate of the PLLA/SSA film for each plasma treatment condition with respect to the porous film cell culture surface manufactured according to an embodiment of the present invention.
- Figure 12A is human hepatic adenocarcinoma cell line , sk-hep1 cells), and
- FIG. 12B is a result of human skin fibroblast (CCD-986sk).
- FIG. 13 shows a schematic diagram of a silicone mold for producing a hydrogel according to an embodiment of the present invention.
- the hydrogels (C and F) fabricated through the mold contain gelatin and lyophilized mesenchymal stem cell culture medium.
- FIG. 14 shows a schematic diagram of a biopatch-type cell therapy product according to an embodiment of the present invention. It is composed of stem cell-cultured film (A), hydrogel (B), and adhesive (Backing layer, C).
- 17 is a result of confirming cell activity and viability according to the presence or absence of the biopatch-type cell therapy agent of the present invention according to an embodiment of the present invention.
- FIG. 18 is a diagram showing a biopatch-type stem cell treatment implantation process in order to evaluate the skin treatment efficacy and effectiveness of the biopatch-type stem cell treatment according to an embodiment of the present invention.
- Figure 19 is a confirmation of the change in the wound area over time for the damaged skin area to which the biopatch-type stem cell therapeutic agent of the present invention has been transplanted according to an embodiment of the present invention
- Figure 19A is a photograph of measuring the wound area
- 19B is the result obtained by deriving the wound area after the passage of time as a percentage compared to the wound area.
- Figure 20 confirms the skin regeneration effect on the damaged skin area to which the biopatch-type stem cell therapeutic agent of the present invention has been transplanted according to an embodiment of the present invention
- Figure 20A is the result of histological analysis through H&E tissue staining of the damaged skin
- Figure 20B is a result of digitizing the length from the normal skin tissue end (Skin margin) to the re-epithelialized part in the tissue staining photograph.
- the inventors of the present invention devised a bandage-type treatment that was applied directly to damaged skin by culturing placenta-derived stem cells on a biodegradable polymer film, and damaged skin using the same. Through treatment, the regenerative ability of the therapeutic agent was confirmed.
- the present invention provides placenta-derived stem cells; It relates to a biopatch-type cell therapy product comprising a porous biodegradable polymer film and a biocompatible polymer scaffold.
- the present invention relates to a biopatch-type cell therapy product in which placenta-derived stem cells are cultured.
- stem cell refers to a master cell capable of regenerating without limitation to form specialized cells of tissues and organs.
- Stem cells are pluripotent or multipotent cells capable of development. Stem cells can divide into two daughter stem cells, or one daughter stem cell and one originating ('transit') cell, which then proliferates into the mature and complete cell form of the tissue.
- placenta-derived stem cells refers to stem cells that can be obtained from the placenta. Specifically, it includes placental stem cells, multipotent cells and progenitor cells.
- pluripotent cell in the present invention refers to a cell that has the ability to grow into any subset of about 260 cell types in the mammalian body. Unlike pluripotent cells, pluripotent cells do not have the ability to form all cell types.
- progenitor cell refers to a cell differentiated into a specific type of cell or adapted to form a specific type of tissue.
- differentiation refers to a phenomenon in which structures and functions are specialized from each other while cells divide and grow, that is, cells, tissues, etc. of organisms change in form or function to perform a given task.
- the "bio patch” is in the form of a thin sheet or film, preferably has a thin film shape, and is attached to any part of the human body such as skin or human organs to activate biological functions and to prevent cell proliferation or differentiation. It means a patch that has biological, therapeutic, and medical functions so that it is possible.
- the main component of the patch is that the cells are cultured and the patch material is biodegradable, so it has a form that is decomposed and disappears in the human body.
- cell therapy product refers to cells and tissues manufactured from human beings through isolation, culture, and special chewing, and is a drug used for the purpose of treatment, diagnosis, and prevention (according to US FDA regulations), which is used to restore the function of cells or tissues. It refers to medicines used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferating and selecting living autologous, allogeneic, or heterogeneous cells in vitro or changing the biological characteristics of cells in other ways.
- Cell therapy agents are largely classified into somatic cell therapy agents and stem cell therapy agents according to the degree of cell differentiation, and the present invention particularly relates to stem cell therapy agents.
- the placenta-derived stem cells may be obtained by an isolation method comprising the following steps.
- haeses and animals refer to mammals belonging to the family Equidae (Genus Eauus ), including horses, donkeys, mules, Grevy's zebras, mountain zebras, quagas of the genus Zebra, Asian wild asses, and African wild asses. , Keng donkeys, tarpans, Przewalski horses, Syrian donkeys, or hybrids born from their crossing belong to this category.
- placenta is made for a fetus during pregnancy, one side of the placenta is in contact with the mother and the other side is in contact with the fetus, and the space between them contains the mother's blood to supply nutrients to the fetus.
- the placenta is composed of three layers: amnion, serous membrane, and decidua.
- amniotic membrane is one layer constituting the placenta in a three-layered structure together with chorion and decidua. It is a thin avascular membrane with a bilayer structure of epithelial monolayer and stromal membrane.
- the placenta-derived stem cells of the present invention obtained by the above isolation method show positive immunological characteristics for mesenchymal stem cell markers CD44 , CD90 , and CD105 , and at the same time, immune cell markers CD34, CD38, CD45, and immune-related markers. It shows negative immunological characteristics for MHC class II .
- the placenta-derived stem cells of the present invention obtained by the above isolation method are pluripotent genes (Oct4, c-Myc, and Klf4) and stem cell-specific genes (PAX6, ALCAM, Integrin- ⁇ 1, Endoglin, and HCAM) show positive genetic expression characteristics.
- placenta-derived stem cells of the present invention obtained by the above isolation method exhibit morphological characteristics of a spindle-shape.
- placenta-derived stem cells of the present invention obtained by the above separation method have the ability to differentiate into cells derived from ectoderm, mesoderm or endoderm.
- placenta-derived stem cells of the present invention obtained by the above separation method have the ability to self-proliferate and be cultured for more than 50 passages while maintaining the characteristics of stem cells.
- the ectoderm-derived cells may be skin cells, nerve cells, and/or astrocytes, the mesoderm-derived cells may be adipocytes, osteoblasts, chondrocytes, and/or muscle cells, and the endoderm-derived cells may be pancreatic cells, and/or lung cells. It may be, but is not limited thereto.
- the present invention relates to a biopatch-type cell therapy product in which placenta-derived stem cells are cultured on the surface of a porous biodegradable polymer film.
- porous means having a property that allows air, moisture, or cells to pass through, and includes, for example, a sheet, film, thin film, or the like in which small through holes are formed.
- the form itself such as a film or a thin film, includes a porous form.
- the porous biodegradable polymer film may be formed by punching a plurality of circular or polygonal holes having a diameter of 1 to 10 ⁇ m.
- 'many' means that the surface area of the entire perforated hole occupies a range of 20 to 50% of the total film area.
- biodegradability means a property that is slowly decomposed without any effect on the human body through biological degradation over time when applied to the human body.
- the surface of the porous biodegradable polymer film may be subjected to sonication and/or plasma treatment.
- the surface of the porous biodegradable polymer film can be treated with ultrasound and/or plasma to induce cell adhesion suitable for culturing stem cells through hydrophilization of the surface.
- a release film may be attached to the back surface of the porous biodegradable polymer film. Specifically, the release film may be included as the outermost layer on the back side of the porous biodegradable polymer film.
- a fluorine-coated polyethylene terephthalate (PET) film may be used, but is not limited thereto.
- the porous biodegradable polymer film is polylactic acid (Poly (lactic acid)), polyglycolic acid (Poly (glycolic acid)), polylactic-glycolic acid (Poly (latic-co-glycolic acid)), polydimethylsiloxane ((Poly )dimethylsiloxane), cellulose, polyhydroxybutyrate hydroxyvalerate (Poly (hydroxybutyrate hydroxyvalerate)), polyethylene glycol and / or poly- ⁇ -caprolactone (Poly- ⁇ -caprolactone) film, It is not limited thereto.
- the polymer film has an amide bond, an ester bond, or a glycosidic bond in the molecular structure of the polymer, so it is biodegradable, and it has a nature-friendly advantage because it is degraded in nature within a few weeks to a few months as short as possible.
- the present invention relates to a biopatch-type cell therapy agent in which a biocompatible polymer scaffold (hydrogel) is attached to the back of a porous biodegradable polymer film in which placenta-derived stem cells are cultured.
- a biocompatible polymer scaffold hydrogel
- the biocompatible polymer scaffold may contain nutrients for cell culture.
- nutrients for cell culture may include, but are not limited to, an extracellular matrix.
- the biocompatible polymer scaffold is attached to the back of the porous biodegradable polymer film in which placenta-derived stem cells are cultured, and nutrients for cell culture (including stem cell culture medium) are supplied to the cells through pores of the porous biodegradable polymer film. By supplying it, it can function to maintain cell activity and maximize the effect of regeneration (treatment) on the affected area. In addition, the role of containing the exudate from the affected area can also be performed incidentally.
- the biocompatible polymer scaffold refers to a structure in the form of a thin film having a certain thickness made of a biocompatible polymer, and has the advantage of being cut into a desired shape for use.
- the biocompatible polymer scaffold may have a thickness of 0.05 to 10.0 mm, but is not limited thereto.
- the biocompatible polymer scaffold may be gelatin, hyaluronic acid, heparin, cellulose, dextran, alginate, chitosan, chitin, collagen, chondroitin sulfate, and/or pectin scaffold, but is not limited thereto.
- the biopatch-type cell therapy product of the present invention may have a form in which placenta-derived stem cells are cultured and a biocompatible polymer scaffold is laminated on a porous biodegradable polymer film.
- FIG. 14 shows a laminated structure of a biopatch-type cell therapy product according to an embodiment of the present invention.
- the biopatch-type cell therapy product of the present invention is composed of a porous biodegradable film in which placenta-derived stem cells are cultured and a biocompatible polymer scaffold (hydrogel) containing nutrients for cell culture. It can be.
- a skin regeneration method using the biopatch-type cell therapy agent of the present invention will be described as follows.
- the biopatch in which the porous biodegradable polymer film in which placenta-derived stem cells are cultured (A) and the biocompatible polymer scaffold (hydrogel) (B) are laminated is cut into an appropriate size or applied to the site without cutting.
- the application site various application sites such as, for example, a burn patient treatment site may be considered.
- the process of attaching to the application site in the process of attaching to the application site, it can be applied in the form of attachment to the application site (skin) by additionally attaching the adhesive (C) to the biocompatible polymer support.
- the stem cells cultured on the surface of the porous biodegradable polymer film move downward along the perforated hole, engraft to the skin surface, and grow while growing. It can exert an effective skin tissue regeneration activity. After a considerable period of time, the porous biodegradable polymer film decomposes and disappears, and the regenerative effect due to the established cells is expressed.
- the present invention relates to a biopatch-type cell therapy product for skin regeneration containing placenta-derived stem cells, which can be transplanted into a living body such as the skin in a state where placenta-derived stem cells are present in a biodegradable polymer film, so contamination or manipulation is complicated. There is an effect that it can be used as a living body transplant material easily without this.
- Figure 1 is a state of transfer of equine amnion tissue to a sterile culture medium.
- Figure 2 shows the results of initial culture of the amnion-derived cells of the present invention according to one embodiment of the present invention.
- Figure 3 confirms the ability of self-replication and proliferation of the amniotic membrane-derived stem cells of the present invention according to an embodiment of the present invention. This is a graph comparing the doubling time of stem cells and skin-derived fibroblasts.
- A before differentiation induction (X100), B: after 7 days of culture (X100), C: after 7 days of culture (X200))
- FIG. 6 is a result of confirming the differentiation ability of the amniotic membrane-derived stem cells of the present invention into osteoblasts according to an embodiment of the present invention.
- A before differentiation induction (X100), B: after 7 days of culture (X100), C: after 7 days of culture (X200))
- RT-PCR reverse transcription polymerase chain reaction
- FIG 11 shows the shape of a porous film cell culture surface manufactured according to an embodiment of the present invention.
- Figure 12 confirms the cell engraftment rate of the PLLA/SSA film for each plasma treatment condition with respect to the porous film cell culture surface manufactured according to an embodiment of the present invention.
- Figure 12A is human hepatic adenocarcinoma cell line , sk-hep1 cells), and
- FIG. 12B is a result of human skin fibroblast (CCD-986sk).
- FIG. 13 shows a schematic diagram of a silicone mold for producing a hydrogel according to an embodiment of the present invention.
- the hydrogels (C and F) fabricated through the mold contain gelatin and lyophilized mesenchymal stem cell culture medium.
- FIG. 14 shows a schematic diagram of a biopatch-type cell therapy product according to an embodiment of the present invention. It is composed of stem cell-cultured film (A), hydrogel (B), and adhesive (Backing layer, C).
- 17 is a result of confirming cell activity and viability according to the presence or absence of the biopatch-type cell therapy agent of the present invention according to an embodiment of the present invention.
- FIG. 18 is a diagram showing a biopatch-type stem cell treatment implantation process in order to evaluate the skin treatment efficacy and effectiveness of the biopatch-type stem cell treatment according to an embodiment of the present invention.
- Figure 19 is a confirmation of the change in the wound area over time for the damaged skin area to which the biopatch-type stem cell therapeutic agent of the present invention has been transplanted according to an embodiment of the present invention
- Figure 19A is a photograph of measuring the wound area
- 19B is the result obtained by deriving the wound area after the passage of time as a percentage compared to the wound area.
- Figure 20 confirms the skin regeneration effect on the damaged skin area to which the biopatch-type stem cell therapeutic agent of the present invention has been transplanted according to an embodiment of the present invention
- Figure 20A is the result of histological analysis through H&E tissue staining of the damaged skin
- Figure 20B is a result of digitizing the length from the normal skin tissue end (Skin margin) to the re-epithelialized part in the tissue staining photograph.
- the present invention relates to a biopatch-type cell therapy product comprising placenta-derived stem cells, a porous biodegradable polymer film, and a biocompatible polymer scaffold.
- the amnion tissue that was separated from the mother and surrounding the foal was transferred to a sterile tub using a sterilized surgical tool, and then transported to the laboratory within an average of about 3 hours while maintaining about 4 ° C.
- the transported amniotic tissue was sterilized once by soaking in 70% ethanol in a UV sterilized aseptic workbench, and then washed three or more times with PBS (phosphate buffered saline) containing 5% penicillin/streptomycin.
- PBS phosphate buffered saline
- DMEM diulbecco Modified Eagle Medium
- FBS fetal bovine serum
- Glutamax fetal bovine serum
- penicillin/streptomycin penicillin/streptomycin
- the culture medium was replaced within 48 h of the initial culture, and the culture medium was also replaced when cells were observed to adhere to the bottom of the culture dish, and passage was performed when more than 80% of the cells attached to the bottom of the culture medium or frozen and stored.
- amnion-derived cells could be observed in an attached form after an average of about 5 days, and as can be seen in FIG. 2, the shape of the cells was observed in a spindle shape similar to that of fibroblasts (FIG. 2A). , which is similar to that seen in other mesenchymal stem cells. Additionally, for detailed observation of cell morphology (X100), it was confirmed by staining with Diff-quik (FIG. 2B).
- Example 1 The amniotic membrane-derived stem cells stored in Example 1 were dispensed at a density of 200,000 cells/well in a 6-well culture dish, and then placed in an incubator at 38.5 °C, 5% CO 2 and cultured without adding any new medium. Incubation time and total number of active cells were observed. As a control, skin-derived fibroblasts were used.
- Doubling time analysis is expressed as an index representing the cell proliferation rate through the analysis of the time at which cells double from the initial number, and is expressed in the following formula.
- N 0 number of cells initially seeded
- N number of final cultured cells
- Viable cells were counted using trypan blue staining. Specifically, the medium in the 6-well culture dish was removed, washed with PBS, and cells were detached from the basal surface using 0.05% Typsin-EDTA. Then, the action of Trypsin was stopped with a culture medium containing 5% FBS, and the cells separated from the cell culture dish were counted.
- the doubling time of the amniotic membrane-derived stem cells (AM-MSC) of the present invention was lower than that of skin-derived fibroblasts (Skin cells) having no therapeutic ability. This indicates that the time point at which the cultured cells divide and double the number initially cultured is faster, suggesting the excellent self-replication and proliferation ability of the amniotic membrane-derived stem cells of the present invention.
- the colony number and size of the amnion-derived stem cells (Equine AM) of the present invention were superior to those of skin-derived fibroblasts having no therapeutic ability. This suggests the excellent self-renewal ability of the amniotic membrane-derived stem cells of the present invention.
- a hanging drop was performed at a concentration of 1,000 cells/ ⁇ L, and cells were placed in an incubator at 38.5° C. and 5% CO 2 to grow in a spherical shape and cultured. Medium was added or exchanged every 3 days. Differentiation was confirmed by staining with alcian blue.
- FIG. 7 staining of amnion stem cells was negative before induction of differentiation (X100, FIG. 7A), but after about 14 days of culture, staining due to chondrocyte differentiation was observed ( X100, Fig. 7B; X200, Fig. 7C).
- CD44, CD90, CD105 showing positive expression in mesenchymal stem cells of Equidae animals presented by the International Society for Cellular Therapy (ISCT), CD34, CD38, CD45 showing negative expression, and immune-related Analysis for the marker MHC class II was performed.
- the expression patterns at the mRNA level were analyzed for embryonic stem cell related genes Oct4, c-Myc, Klf4 and mesenchymal stem cell related genes PAX6, ALCAM, Integrin- ⁇ 1, Endoglin, and HCAM, known as characteristics of amniotic membrane-derived stem cells. did
- RNA was prepared using an RNA extraction kit according to the manufacturer's instructions (Qiagen, Cat. No. 74104). For RNA samples (1 ⁇ l of total RNA), cDNA was synthesized with oligo dT primers and random primers using iScript reverse transcriptase (BIO-RAD). Specific primer sequences are shown in Table 1 below.
- RT-PCR was denatured at 95 °C for 4 minutes, then reacted 34 times at 95 °C for 30 seconds, 56 °C for 30 seconds, and 72 °C for 30 seconds. A final extension was performed at 73 °C for 5 minutes.
- Beta-Actin RT-PCR reaction was performed by modifying the primer annealing at 34 times and 60 °C. RT-PCR specific amplification of the target gene was confirmed by DNA sequencing. As a result, as shown in FIG. 8, embryonic stem cell-related genes (Oct4, c-Myc, and Klf4) and mesenchymal stem cell-related genes (PAX6, ALCAM, Integrin-beta1, Endoglin, and HCAM) were all expressed. 2) Quantitative real time PCR (qRT-PCR)
- cDNA was synthesized by the same method as the cDNA synthesis method used for the RT-PCR. qRT-PCR was denatured at 95 °C for 10 minutes, followed by 39 reactions at 94 °C for 15 seconds, 58 °C for 30 seconds, and 72 °C for 30 seconds. After reacting at 95 °C for 10 seconds and 65 °C for 5 seconds, the amount of marker expression was compared with that of control horse skin-derived fibroblasts.
- genes (Oct4, c-Myc, and Klf4) exhibiting pluripotency in amnion-derived stem cells (AM-MSC) of the present invention compared to skin-derived fibroblasts (Skin) having no therapeutic ability.
- A-MSC amnion-derived stem cells
- Skin skin-derived fibroblasts
- stem cell-specific genes PAX6, ALCAM, Integrin-beta1, Endoglin, and HCAM
- FITC fluorescein isothiocyanate
- PE phytoerythrin
- the antibodies used for comparison of relative expression patterns based on secondary IgG antibodies are as follows: CD29-PE, CD34-FITC, CD38-FITC, CD44-FITC, CD45, CD90-PE, CD105-FITC, MHC class II -FITC and IgG-FITC. About 5-8x10 5 cells per sample were used to analyze one antibody, and flow cytometry was performed using Cellquest software (FACScan, BD Biosciences, USA).
- a biodegradable poly(L-Lactic acid) (PLLA) film was prepared with a size of 15 x 15 (cm). First, one side was sealed using clean paper to protect the cell culture side during the adhesive coating process to be described later. Then, for affinity between the adhesive and the PLLA film, a primer was treated on the opposite side of the cell culture side of the PLLA film. Specifically, after soaking cotton with DY 39-067 Rubber Additive (SILASTIC) and evenly treating it once on the PLLA film, it was dried in an oven at 40 ° C for 5 minutes, and the solvent was blown off sufficiently.
- SILASTIC DY 39-067 Rubber Additive
- Two-component silicone adhesive, 7-9700 Soft Skin Adhesive (DOW CORNING ® ) were mixed in a ratio of 45:55 and stirred for 2 minutes at 1000 rpm using an overhead stirrer. Then, after confirming that bubbles had sufficiently settled, coating was performed using an automatic coating machine (Ocean science) and a baker applicator. Coating was performed at a speed of 50 mm/sec with a baker applicator set to 50 ⁇ m after placing 3 g of adhesive based on a 15 x 15 (cm) film on the film. The adhesive-coated film was cured in an oven at 40 °C for 6 hours.
- a fluorine-treated PET (polyethylene terephthalate) release film (FL-25BMM, Giseung Industry) was attached using a semi-automatic mangle (Ocean science).
- the adhesive-coated PLLA film and the fluorine-treated PET release film were placed between the rolls of the fader mangle, and the height was adjusted by adjusting the handle. The roll speed was 10 rpm.
- a precision perforation machine was manufactured in-house to produce a porous film in a way to minimize warpage of the PLLA film attached with the PET release film.
- the diameter of one hole in the porous film was about 1.5 microns, and the total area of the hole in the porous film accounted for 20 to 50% of the total area of the film (see FIG. 11).
- Cell culture was designed to enable cell culture on a biodegradable film made of transparent polymer material through the design of the cell culture surface in a way that is advantageous to the post-process (meaning the process after perforation).
- the cell engraftment rate test of the PLLA/SSA (Soft skin adhesive) film was conducted for each plasma treatment condition. Confirmation of cell engraftment and viability of human hepatic adenocarcinoma cell line (sk-hep1 cells) (ATCC) and human skin fibroblast (CCD-986sk) (ATCC) under various plasma conditions did
- Plate A was used as a negative and positive control, respectively, without cells (-) or with cells (+) without a film attached to a general cell culture plate.
- Plate B was used as another control with the same polystyrene film as Plate A.
- Plate C-F is a cell culture complex (PLLA/SSA) treated with different types of plasma. Specific argon (lpm) / oxygen (sccm) gas conditions are shown in Table 2 below.
- dialysis was performed using 0.1 M NaCl solution, 25% ethanol, and tertiary distilled water, and the dialysis solution was lyophilized to prepare a gelatin sponge modified with tyramine.
- Gelatin modified with tyramine was dissolved in tertiary distilled water at 6wt% and 35 °C.
- lyophilized powder of 10 ml cell culture medium was dissolved in 1 ml of tertiary distilled water at 4 °C.
- a biopatch was fabricated by sequentially arranging the porous film for stem cell culture prepared above (FIG. 14A), the hydrogel containing the cell culture medium (FIG. 14B), and the skin adhesive (FIG. 14C). .
- Amniotic membrane-derived cells between passages 3 and 5 were cultured using DMEM culture medium containing 15% FBS, 1% Glutamax, and 1% penicillin/streptomycin. After removing the culture medium in the culture dish and washing with PBS, the cells were detached from the basal surface using 0.05% Typsin-EDTA. The separated cells were cultured at 1x10 4 cells/cm 2 number.
- Stem cells cultured for 48 hours in a culture dish without a biopatch and a culture dish with a biopatch prepared in Example 5 were washed with PBS, separated from the basal surface using 0.05% Typsin-EDTA, and centrifuged. Cells were collected using a separator. After staining the collected cells with trypan blue, the stained cells were coated on a hemocytometer and observed under an optical microscope. The number and viability were determined by counting the number of unstained and stained cells.
- the cells After washing the cells cultured for 2 days in a culture dish without a biopatch and a culture dish with a biopatch prepared in Example 5 with PBS, the cells were separated from the basal surface using 0.05% Trpsin-EDTA and centrifuged. The cells were collected using The cells were stained using a kit (No. V13241, Invitrogen) and analyzed for viable cells, apoptosis and necrosis using a flow cytometer.
- a kit No. V13241, Invitrogen
- mice For 6-week-old male ICR mice (Doo-yeol Biotech, KOREA), efficacy evaluation was conducted in accordance with the establishment and operation regulations of the Animal Experimentation Ethics Committee of Chungnam National University (approval number, 202103A-CNU-072). The mice were brought in at 5 weeks of age, stabilized for one week, anesthetized by intraperitoneal administration of an anesthetic (80 mg/kg ketamine + 12 mg/kg xylazine), and wounds were induced through a biopsy punch.
- an anesthetic 80 mg/kg ketamine + 12 mg/kg xylazine
- the wound area was measured at 0, 3, 7, 10, or 14 days after transplantation of the therapeutic agent (FIG. 19a), and the change in wound area was confirmed using the Image J program. (Fig. 19b).
- skin tissue of the wound portion was collected 3 or 7 days after transplantation of the therapeutic agent. After fixing the collected tissue with a 10% formalin solution, a paraffin block was prepared. Hematoxylin and eosin (H&E) staining of slides cut to a thickness of 4 ⁇ m from the prepared paraffin block was performed to evaluate granulation tissue formation (Fig. 20a) and skin re-epithelialization (Fig. 20b). The skin re-epithelialization was quantified by using the Image J program as the length from the skin margin to the re-epithelialized area.
- H&E Hematoxylin and eosin
- the present invention relates to a biopatch-type cell therapy product for skin regeneration containing placenta-derived stem cells.
- Bio-patch type cell therapy product for Regenerating skin comprising Placenta-derived stem cells
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Abstract
Description
본 발명은 태반-유래 줄기세포를 함유하는 피부 재생용 바이오패치형 세포치료제에 관한 것이다.The present invention relates to a biopatch-type cell therapy product for skin regeneration containing placenta-derived stem cells.
줄기세포(stem cell)는 자기 복제가 가능하고 한 세포에서 다양한 조직을 구성하는 세포로 분화 및 증식이 가능 아직 분화되지 않은 미분화 세포로서, 손상 받은 신체 조직 및 세포를 재생할 수 있는 특이한 능력을 보유하고 있다. 현재 확실한 치료법이 없는 퇴행성 질병이나 심한 외상과 같은 치료가 어려운 손상에 있어 의학적 활용도가 매우 높은 차세대 기술로 인식되어 알려져 있으며, 재생의학 분야에서 줄기세포를 이용한 임상 시험 및 연구가 진행되고 있다. Stem cells are undifferentiated cells that are capable of self-replication and can differentiate and proliferate from one cell into cells constituting various tissues. They have a unique ability to regenerate damaged body tissues and cells. there is. It is recognized and known as a next-generation technology with high medical applicability for difficult-to-treat injuries such as degenerative diseases for which there is currently no definite treatment or severe trauma, and clinical trials and research using stem cells are being conducted in the field of regenerative medicine.
줄기세포는 세포 기원 조직의 발달 상태에 따라 크게 배아 유래 줄기세포(embryonic stem cells)와 성체 줄기세포(adult stem cells)로 구분되며, 그 외에 유전자 편집기술을 이용한 만능 유도 줄기세포(induced Pluripotent stem cells)가 있다. 줄기세포를 이용한 재생 치료 연구는 조직 원료 획득의 용이성, 줄기세포 분리 효율 및 종양 형성 문제 등을 고려하여 주로 성체 줄기세포의 한 종류인 중간엽 줄기세포(mesenchymal stem cells)가 이용되고 있으며, 골수, 지방, 태반 등의 조직에서 얻을 수 있다.Stem cells are largely classified into embryonic stem cells and adult stem cells according to the developmental state of the tissue of origin. In addition, induced pluripotent stem cells using gene editing technology ) is there. Regenerative treatment research using stem cells mainly uses mesenchymal stem cells, a type of adult stem cells, in consideration of the ease of obtaining tissue raw materials, the efficiency of stem cell isolation, and the problem of tumor formation. It can be obtained from tissues such as fat and placenta.
태반 줄기세포는 제대혈, 양막 및 양수에서 유래한 줄기세포로, 다른 조직 유래 중간엽 줄기세포 중에서 가장 최근에 발견되어 연구되고 있다. 태반 줄기세포는 우수한 증식 능력을 가지고 있으며 종양형성 문제가 없고, 지방, 뼈, 근육, 간, 신경 등의 중배엽, 내배엽, 외배엽 기원 조직으로 분화할 수 있는 능력을 지니고 있다. 특히 윤리적인 문제를 극복한 유일한 줄기세포이며, 출생 시 비침습적인 방법으로 확보가 가능한 장점이 있으며, 위와 같은 결과들은 성체 줄기세포 기원 조직 중 태아와 함께 생성되어 가장 빨리 채취 가능한 조직으로 태반 줄기세포의 세포 치료제로서의 가능성을 보여준다.Placental stem cells are stem cells derived from umbilical cord blood, amnion, and amniotic fluid, and have been recently discovered and studied among other tissue-derived mesenchymal stem cells. Placental stem cells have excellent proliferative ability, have no tumor formation problem, and have the ability to differentiate into mesodermal, endodermal, and ectodermal tissues such as fat, bone, muscle, liver, and nerve. In particular, it is the only stem cell that has overcome ethical problems, and has the advantage of being non-invasively obtained at birth. shows potential as a cell therapy.
하지만 줄기세포를 이용한 재생 치료는 이식한 줄기세포의 생존율 저하로 인한 이식 및 전달 효율성이 제한된다는 문제가 있으며, 최근 여러 기술과의 융합을 통하여 세포 단일 치료제 개발에 집중되어 왔던 연구 방향에서 안정성과 효용성이 보완된 전략 기술 발달이 진행되고 있다. However, regenerative treatment using stem cells has a problem in that the efficiency of transplantation and delivery is limited due to the decrease in the viability of transplanted stem cells, and stability and effectiveness in research direction that has been focused on the development of single cell therapy through convergence with various technologies This complementary strategic technology development is ongoing.
한편, 바이오패치(Bio-patch)는 일반적으로 수술, 주사 및 피부 상처로 야기되는 감염을 예방하기 위한 목적으로 피부에 부착하는 일종의 드레싱으로서 사용되며, 최근 바이오 패치 기술은 건강관련 생체신호를 실시간 분석, 감지하는 패치형 센서, 만성질환 및 건강을 관리하는 부착형 생체신호 모니터링 모듈로도 응용되어 연구 개발되고 있다.On the other hand, a bio-patch is generally used as a kind of dressing attached to the skin for the purpose of preventing infections caused by surgery, injection, and skin wounds, and recent bio-patch technology analyzes health-related biosignals in real time. It is also being researched and developed as a patch-type sensor that detects blood pressure, and an attachable bio-signal monitoring module that manages chronic diseases and health.
도 1은 말의 양막 조직을 멸균된 배양 배지에 옮긴 모습이다.Figure 1 is a state of transfer of equine amnion tissue to a sterile culture medium.
도 2는 본 발명의 일 실시예에 따라, 본 발명의 양막 유래 세포의 초기 배양 결과를 나타낸다.Figure 2 shows the results of initial culture of the amnion-derived cells of the present invention according to one embodiment of the present invention.
도 3은 본 발명의 일 실시예에 따라, 본 발명의 양막 유래 줄기세포의 자가 복제 및 증식 능력을 확인한 것으로, 도 3A는 초기 계대부터 45 계대 이상의 양막 유래 줄기세포의 Doubling time, 도 3B는 양막 유래 줄기세포와 피부 유래 섬유아세포의 doubling time을 비교한 그래프이다. Figure 3 confirms the ability of self-replication and proliferation of the amniotic membrane-derived stem cells of the present invention according to an embodiment of the present invention. This is a graph comparing the doubling time of stem cells and skin-derived fibroblasts.
도 4는 양막 유래 줄기세포와 피부 유래 섬유아세포의 콜로니 형성 능력을 비교한 결과이다.4 is a result of comparing the colony formation abilities of amnion-derived stem cells and skin-derived fibroblasts.
도 5는 본 발명의 일 실시예에 따라, 본 발명의 양막 유래 줄기세포의 지방세포로의 분화 능력을 확인한 결과이다. (A: 분화 유도 전(X100), B: 배양 7일 후(X100), C: 배양 7일 후(X200))5 is a result of confirming the differentiation ability of amniotic membrane-derived stem cells of the present invention into adipocytes according to an embodiment of the present invention. (A: before differentiation induction (X100), B: after 7 days of culture (X100), C: after 7 days of culture (X200))
도 6은 본 발명의 일 실시예에 따라, 본 발명의 양막 유래 줄기세포의 골아세포로의 분화 능력을 확인한 결과이다. (A: 분화 유도 전(X100), B: 배양 7일 후(X100), C: 배양 7일 후(X200))6 is a result of confirming the differentiation ability of the amniotic membrane-derived stem cells of the present invention into osteoblasts according to an embodiment of the present invention. (A: before differentiation induction (X100), B: after 7 days of culture (X100), C: after 7 days of culture (X200))
도 7은 본 발명의 일 실시예에 따라, 본 발명의 양막 유래 줄기세포의 연골세포로의 분화 능력을 확인한 결과이다. (A: 분화 유도 전(X100), B: 배양 7일 후(X100), C: 배양 7일 후(X200))7 is a result of confirming the differentiation ability of amniotic membrane-derived stem cells of the present invention into chondrocytes according to an embodiment of the present invention. (A: before differentiation induction (X100), B: after 7 days of culture (X100), C: after 7 days of culture (X200))
도 8은 본 발명의 일 실시예에 따라, 역전사 중합효소 연쇄반응(RT-PCR)을 이용하여 본 발명의 양막 유래 줄기세포의 다능성을 확인한 결과이다. (컨트롤 마커: beta-actin)8 is a result of confirming the pluripotency of amniotic membrane-derived stem cells of the present invention using reverse transcription polymerase chain reaction (RT-PCR) according to an embodiment of the present invention. (Control marker: beta-actin)
도 9는 본 발명의 일 실시예에 따라, 실시간 정량(qRT-PCR)을 이용하여 본 발명의 양막 유래 줄기세포의 다능성을 확인한 결과이다. 9 is a result of confirming the pluripotency of the amniotic membrane-derived stem cells of the present invention using real-time quantitative (qRT-PCR) according to an embodiment of the present invention.
도 10은 본 발명의 일 실시예에 따라, 유세포 분석을 이용하여 본 발명의 양막 유래 줄기세포의 다능성을 확인한 결과이다. (red line: negative control, blue line: 양성 발현 데이터)10 is a result of confirming the pluripotency of the amniotic membrane-derived stem cells of the present invention using flow cytometry according to an embodiment of the present invention. (red line: negative control, blue line: positive expression data)
도 11은 본 발명의 일 실시예에 따라 제작된 다공성 필름 세포 배양면의 형태를 나타낸다. 11 shows the shape of a porous film cell culture surface manufactured according to an embodiment of the present invention.
도 12는 본 발명의 일 실시예에 따라 제작된 다공성 필름 세포 배양면에 대하여, 플라즈마 처리 조건별 PLLA/SSA 필름의 세포 생착률을 확인한 것으로, 도 12A는 인간 혈관내피 유래 암세포(Human hepatic adenocarcinoma cell line, sk-hep1 cells), 도 12B는 인간 피부 유래 섬유아세포(Human skin Fibroblast, CCD-986sk)에 대한 결과이다.Figure 12 confirms the cell engraftment rate of the PLLA/SSA film for each plasma treatment condition with respect to the porous film cell culture surface manufactured according to an embodiment of the present invention. Figure 12A is human hepatic adenocarcinoma cell line , sk-hep1 cells), and FIG. 12B is a result of human skin fibroblast (CCD-986sk).
도 13은 본 발명의 일 실시예에 따른 하이드로젤 제작용 실리콘 몰드의 모식도를 나타낸다. 몰드를 통해 제작된 하이드로젤(C 및 F)은 젤라틴과 동결건조한 중간엽 줄기세포 배양액을 함유한다.13 shows a schematic diagram of a silicone mold for producing a hydrogel according to an embodiment of the present invention. The hydrogels (C and F) fabricated through the mold contain gelatin and lyophilized mesenchymal stem cell culture medium.
도 14는 본 발명의 일 실시예에 따른 바이오패치형 세포치료제의 모식도를 나타낸다. 줄기세포가 배양된 필름(A), 하이드로젤(B), 점착제(Backing layer, C)로 구성된다. 14 shows a schematic diagram of a biopatch-type cell therapy product according to an embodiment of the present invention. It is composed of stem cell-cultured film (A), hydrogel (B), and adhesive (Backing layer, C).
도 15는 본 발명의 일 실시예에 따라, 본 발명의 바이오패치형 세포치료제의 유무에 따른 세포 형태를 확인한 결과이다. (A: 바이오패치가 없는 경우, B: 바이오패치에 세포를 배양한 경우)15 is a result of confirming the cell morphology according to the presence or absence of the biopatch-type cell therapy agent of the present invention according to an embodiment of the present invention. (A: In case of no biopatch, B: In case of culturing cells in biopatch)
도 16은 본 발명의 일 실시예에 따라, 본 발명의 바이오패치형 세포치료제의 유무에 따른 줄기세포 증식 능력을 확인한 결과이다. 16 is a result of confirming stem cell proliferation ability according to the presence or absence of the biopatch-type cell therapy agent of the present invention according to an embodiment of the present invention.
도 17은 본 발명의 일 실시예에 따라, 본 발명의 바이오패치형 세포치료제의 유무에 따른 세포 활성 및 생존 능력을 확인한 결과이다.17 is a result of confirming cell activity and viability according to the presence or absence of the biopatch-type cell therapy agent of the present invention according to an embodiment of the present invention.
도 18은 본 발명의 일 실시예에 따라, 본 발명의 바이오패치형 줄기세포 치료제의 피부 치료 효능 유효성을 평가를 위하여, 바이오패치형 줄기세포 치료제 이식 과정을 나타낸 도이다.18 is a diagram showing a biopatch-type stem cell treatment implantation process in order to evaluate the skin treatment efficacy and effectiveness of the biopatch-type stem cell treatment according to an embodiment of the present invention.
도 19는 본 발명의 일 실시예에 따라, 본 발명의 바이오패치형 줄기세포 치료제가 이식된 피부 손상 부위에 대한 시간 경과에 따른 상처 면적 변화를 확인한 것으로, 도 19A는 상처 면적을 측정한 사진, 도 19B는 상처 면적 대비 시간 경과 후의 상처 면적을 %로 도출하여 나타낸 결과이다.Figure 19 is a confirmation of the change in the wound area over time for the damaged skin area to which the biopatch-type stem cell therapeutic agent of the present invention has been transplanted according to an embodiment of the present invention, and Figure 19A is a photograph of measuring the wound area; 19B is the result obtained by deriving the wound area after the passage of time as a percentage compared to the wound area.
도 20은 본 발명의 일 실시예에 따라, 본 발명의 바이오패치형 줄기세포 치료제가 이식된 피부 손상 부위에 대한 피부 재생 효과를 확인한 것으로, 도 20A는 손상된 피부의 H&E 조직 염색을 통한 조직학적 분석 결과, 도 20B는 조직 염색 사진에서 정상 피부 조직 끝부분(Skin margin)에서부터 재상피화가 진행된 부분까지의 길이를 수치화 하여 나타낸 결과이다.Figure 20 confirms the skin regeneration effect on the damaged skin area to which the biopatch-type stem cell therapeutic agent of the present invention has been transplanted according to an embodiment of the present invention, and Figure 20A is the result of histological analysis through H&E tissue staining of the damaged skin , Figure 20B is a result of digitizing the length from the normal skin tissue end (Skin margin) to the re-epithelialized part in the tissue staining photograph.
본 발명자들은 피부 조직 재생용 바이오패치형 세포치료제를 개발하고자 연구하던 중, 태반-유래 줄기세포를 생분해성 고분자 필름에 배양하여 피부 손상 부위에 바로 적용하는 반창고 타입의 치료제를 고안하고, 이를 이용한 피부 손상 치료를 통하여 치료제의 재생 능력을 확인하였다.While researching to develop a biopatch-type cell therapy product for regenerating skin tissue, the inventors of the present invention devised a bandage-type treatment that was applied directly to damaged skin by culturing placenta-derived stem cells on a biodegradable polymer film, and damaged skin using the same. Through treatment, the regenerative ability of the therapeutic agent was confirmed.
따라서, 본 발명은 태반-유래 줄기세포; 다공성 생분해성 고분자 필름 및 생체적합성 고분자 지지체를 포함하는, 바이오패치형 세포치료제에 관한 것이다.Accordingly, the present invention provides placenta-derived stem cells; It relates to a biopatch-type cell therapy product comprising a porous biodegradable polymer film and a biocompatible polymer scaffold.
이하, 본 발명을 더욱 자세히 설명하고자 한다.Hereinafter, the present invention will be described in more detail.
본 발명의 일 구현예에 따르면, 본 발명은 태반-유래 줄기세포가 배양되어 있는 바이오패치형 세포치료제에 관한 것이다.According to one embodiment of the present invention, the present invention relates to a biopatch-type cell therapy product in which placenta-derived stem cells are cultured.
본 발명에서 "줄기세포"는 조직 및 기관의 특수화된 세포를 형성하도록 비제한적으로 재생할 수 있는 마스터 세포를 지칭한다. 줄기세포는 발달가능한 만능성 또는 다능성 세포이다. 줄기세포는 2개의 딸줄기세포, 또는 하나의 딸줄기세포와 하나의 유래('전이(transit)') 세포로 분열될 수 있으며, 이후에 조직의 성숙하고 완전한 형태의 세포로 증식된다.In the present invention, "stem cell" refers to a master cell capable of regenerating without limitation to form specialized cells of tissues and organs. Stem cells are pluripotent or multipotent cells capable of development. Stem cells can divide into two daughter stem cells, or one daughter stem cell and one originating ('transit') cell, which then proliferates into the mature and complete cell form of the tissue.
본 발명에서 "태반-유래 줄기세포"는 태반으로부터 수득 될 수 있는 줄기세포를 지칭한다. 구체적으로는 태반 줄기세포, 다능성(multipotent) 세포 및 전구(progenitor) 세포를 포함한다.In the present invention, "placenta-derived stem cells" refers to stem cells that can be obtained from the placenta. Specifically, it includes placental stem cells, multipotent cells and progenitor cells.
본 발명에서 "다능성 세포"는 포유류 신체의 약 260개 세포 유형의 임의의 하위세트로의 성장능력을 갖는 세포를 지칭한다. 만능성 세포와 달리, 다능성 세포는 모든 세포 유형을 형성하려는 능력을 갖지 않는다.A "pluripotent cell" in the present invention refers to a cell that has the ability to grow into any subset of about 260 cell types in the mammalian body. Unlike pluripotent cells, pluripotent cells do not have the ability to form all cell types.
본 발명에서 "전구 세포"는 특정 유형의 세포로 분화되거나 특정 유형의 조직을 형성하도록 된 세포를 지칭한다.In the present invention, "progenitor cell" refers to a cell differentiated into a specific type of cell or adapted to form a specific type of tissue.
본 발명에서 "분화"는 세포가 분열 증식하여 성장하는 동안에 서로 구조나 기능이 특수화하는 현상, 즉 생물의 세포, 조직 등이 각각에게 주어진 일을 수행하기 위하여 형태나 기능이 변해가는 것을 말한다.In the present invention, "differentiation" refers to a phenomenon in which structures and functions are specialized from each other while cells divide and grow, that is, cells, tissues, etc. of organisms change in form or function to perform a given task.
본 발명에서 "바이오패치"는 얇은 시트 또는 필름 형상으로서 바람직하게는 박막 형상을 가질 수 있으며, 피부나 인체의 장기 등 인체 부위의 어느 곳이든 부착하여 생물학적 기능을 활성화하고 세포의 증식이나 분화 등이 가능하도록 생물학적, 치료학적, 의료적 기능성을 가진 패치를 의미한다. 본질적으로는 패치의 주성분은 세포가 배양되어 있으면서 패치 재질은 생분해성을 가지므로 인체에서 분해되어 소멸되는 형태를 가진다.In the present invention, the "bio patch" is in the form of a thin sheet or film, preferably has a thin film shape, and is attached to any part of the human body such as skin or human organs to activate biological functions and to prevent cell proliferation or differentiation. It means a patch that has biological, therapeutic, and medical functions so that it is possible. Essentially, the main component of the patch is that the cells are cultured and the patch material is biodegradable, so it has a form that is decomposed and disappears in the human body.
본 발명에서 "세포치료제"는 사람으로부터 분리, 배양 및 특수한 저작을 통해 제조된 세포 및 조직으로 치료, 진단 및 예방의 목적으로 사용되는 의약품(미국 FDA규정)으로서, 세포 혹은 조직의 기능을 복원시키기 위하여 살아있는 자가, 동종, 또는 이종 세포를 체외에서 증식·선별하거나 다른 방법으로 세포의 생물학적 특성을 변화시키는 등의 일련의 행위를 통하여 치료, 진단 및 예방의 목적으로 사용되는 의약품을 지칭한다. 세포치료제는 세포의 분화정도에 따라 크게 체세포치료제, 줄기세포치료제로 분류되며 본 발명은 특히 줄기세포치료제에 관한 것이다.In the present invention, "cell therapy product" refers to cells and tissues manufactured from human beings through isolation, culture, and special chewing, and is a drug used for the purpose of treatment, diagnosis, and prevention (according to US FDA regulations), which is used to restore the function of cells or tissues. It refers to medicines used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferating and selecting living autologous, allogeneic, or heterogeneous cells in vitro or changing the biological characteristics of cells in other ways. Cell therapy agents are largely classified into somatic cell therapy agents and stem cell therapy agents according to the degree of cell differentiation, and the present invention particularly relates to stem cell therapy agents.
상기 태반-유래 줄기세포는 다음의 단계를 포함하는 분리방법에 의해 얻어진 것일 수 있다.The placenta-derived stem cells may be obtained by an isolation method comprising the following steps.
말과 동물 모체의 출산 후 분리되는 태반으로부터 양막(equine amniotic membrane) 조직을 얻는 단계; Obtaining equine amniotic membrane tissue from the placenta separated after childbirth of horses and animal mothers;
양막 조직을 파쇄(chopping)하는 단계;chopping the amnion tissue;
원심분리하여 펠렛(pellet)을 회수하는 단계;Recovering a pellet by centrifugation;
세척 및 원심분리를 반복하여 단핵세포를 분리하는 단계; 및Separating mononuclear cells by repeating washing and centrifugation; and
소태아혈청 및 항생제가 첨가된 배지에서 배양하는 단계.Culturing in a medium supplemented with fetal bovine serum and antibiotics.
본 발명에서 "말과 동물"이란, 말과(Equidae; Genus Eauus)에 속하는 포유류 동물로, 말, 당나귀, 노새, 그레비얼룩말, 산얼룩말, 얼룩말아속의 콰가, 아시아야생당나귀, 아프리카야생당나귀, 컁당나귀, 타르판, 프셰발스키말, 시리아당나귀, 또는 이들의 교잡으로 태어난 잡종 등이 이에 속한다.In the present invention, "horses and animals" refer to mammals belonging to the family Equidae (Genus Eauus ), including horses, donkeys, mules, Grevy's zebras, mountain zebras, quagas of the genus Zebra, Asian wild asses, and African wild asses. , Keng donkeys, tarpans, Przewalski horses, Syrian donkeys, or hybrids born from their crossing belong to this category.
본 발명에서 "태반"은 임신 중에 태아를 위해 만들어지는 것으로, 태반의 한쪽은 모체와 닿아 있고 다른 한쪽은 태아와 맞닿아 있으며 그 사이 공간에 모체의 혈액이 담겨 있어 태아에게 영양분을 공급하게 된다. 태반은 양막, 장막, 탈락막의 3층으로 구성되어 있다.In the present invention, "placenta" is made for a fetus during pregnancy, one side of the placenta is in contact with the mother and the other side is in contact with the fetus, and the space between them contains the mother's blood to supply nutrients to the fetus. The placenta is composed of three layers: amnion, serous membrane, and decidua.
본 발명에서 "양막"은 융모막, 탈락막과 함께 삼중층의 구조로 태반을 구성하는 하나의 층이다. 상피 단층과 기질막의 이중층 구조를 갖는 얇은 무혈관 막으로 태아에 결합하여 환경을 구성하는 주머니이다. In the present invention, "amniotic membrane" is one layer constituting the placenta in a three-layered structure together with chorion and decidua. It is a thin avascular membrane with a bilayer structure of epithelial monolayer and stromal membrane.
상기 분리방법에 의해 얻어진 본 발명의 태반-유래 줄기세포는 중간엽 줄기세포 마커인 CD44, CD90, CD105에 대하여 양성의 면역학적 특성을 나타내고, 동시에 면역 세포 마커인 CD34, CD38, CD45, 면역 관련 마커인 MHC class II에 대하여 음성의 면역학적 특성을 나타낸다.The placenta-derived stem cells of the present invention obtained by the above isolation method show positive immunological characteristics for mesenchymal stem cell markers CD44 , CD90 , and CD105 , and at the same time, immune cell markers CD34, CD38, CD45, and immune-related markers. It shows negative immunological characteristics for MHC class II .
또한, 상기 분리방법에 의해 얻어진 본 발명의 태반-유래 줄기세포는 다능성을 나타내는 유전자(Oct4, c-Myc, 및 Klf4) 및 줄기세포에 특이적으로 나타나는 유전자(PAX6, ALCAM, Integrin-β1, Endoglin, 및 HCAM)에 대하여 모두 양성의 유전적 발현 특성을 나타낸다.In addition, the placenta-derived stem cells of the present invention obtained by the above isolation method are pluripotent genes (Oct4, c-Myc, and Klf4) and stem cell-specific genes (PAX6, ALCAM, Integrin-β1, Endoglin, and HCAM) show positive genetic expression characteristics.
또한, 상기 분리방법에 의해 얻어진 본 발명의 태반-유래 줄기세포는 입방 형태(spindle-shape)의 형태학적 특성을 나타낸다.In addition, the placenta-derived stem cells of the present invention obtained by the above isolation method exhibit morphological characteristics of a spindle-shape.
또한, 상기 분리방법에 의해 얻어진 본 발명의 태반-유래 줄기세포는 외배엽, 중배엽 또는 내배엽 유래 세포로 분화하는 능력을 가진다.In addition, the placenta-derived stem cells of the present invention obtained by the above separation method have the ability to differentiate into cells derived from ectoderm, mesoderm or endoderm.
나아가, 상기 분리방법에 의해 얻어진 본 발명의 태반-유래 줄기세포는 줄기세포의 특성을 유지 한 채 50 계대 이상 배양되는 자가 증식능력을 가진다.Furthermore, the placenta-derived stem cells of the present invention obtained by the above separation method have the ability to self-proliferate and be cultured for more than 50 passages while maintaining the characteristics of stem cells.
상기 외배엽 유래 세포는 피부세포, 신경세포, 및/또는 성상세포, 상기 중배엽 유래 세포는 지방세포, 골아세포, 연골세포, 및/또는 근세포, 상기 내배엽 유래 세포는 췌장세포, 및/또는 폐세포일 수 있으나, 이에 제한되는 것은 아니다.The ectoderm-derived cells may be skin cells, nerve cells, and/or astrocytes, the mesoderm-derived cells may be adipocytes, osteoblasts, chondrocytes, and/or muscle cells, and the endoderm-derived cells may be pancreatic cells, and/or lung cells. It may be, but is not limited thereto.
본 발명의 다른 일 구현예에 따르면, 본 발명은 다공성 생분해성 고분자 필름 표면에 태반-유래 줄기세포가 배양되어 있는 바이오패치형 세포치료제에 관한 것이다.According to another embodiment of the present invention, the present invention relates to a biopatch-type cell therapy product in which placenta-derived stem cells are cultured on the surface of a porous biodegradable polymer film.
본 발명에서 "다공성"은 공기나 습기 또는 세포가 투과할 수 있는 성질을 가진 것을 의미하며, 예컨대 시트나 필름, 박막 등의 형상에 작은 관통 구멍이 형성된 것을 포함한다. 통상적으로는 필름, 박막 등의 형태 자체가 다공질의 형태를 가진 것을 포함한다.In the present invention, "porous" means having a property that allows air, moisture, or cells to pass through, and includes, for example, a sheet, film, thin film, or the like in which small through holes are formed. Usually, the form itself, such as a film or a thin film, includes a porous form.
본 발명의 일 구체예에서, 상기 다공성 생분해성 고분자 필름은 직경 1~10 μm 크기의 원형 또는 다각형상의 구멍이 다수 천공된 형성된 것일 수 있다. 여기서 '다수'라는 의미는 천공된 전체 구멍의 표면적이 전체 필름 면적의 20~50%범위를 차지하는 것을 의미한다.In one embodiment of the present invention, the porous biodegradable polymer film may be formed by punching a plurality of circular or polygonal holes having a diameter of 1 to 10 μm. Here, 'many' means that the surface area of the entire perforated hole occupies a range of 20 to 50% of the total film area.
**본 발명에서 "생분해성"은 인체에 적용 시 시간이 경과함에 따라 생물학적 분해를 통해 인체에 아무런 영향을 주지 아니하고 서서히 분해되는 성질을 의미한다.** In the present invention, "biodegradability" means a property that is slowly decomposed without any effect on the human body through biological degradation over time when applied to the human body.
상기 다공성 생분해성 고분자 필름 표면은 초음파 처리(sonication) 및/또는 플라즈마 처리된 것일 수 있다. 상기 다공성 생분해성 고분자 필름 표면은 초음파 및/또는 플라즈마 처리함으로써 표면 친수화를 통한 줄기세포의 배양에 적합한 세포 부착력이 유도될 수 있다.The surface of the porous biodegradable polymer film may be subjected to sonication and/or plasma treatment. The surface of the porous biodegradable polymer film can be treated with ultrasound and/or plasma to induce cell adhesion suitable for culturing stem cells through hydrophilization of the surface.
상기 다공성 생분해성 고분자 필름 이면은 이형 필름이 부착된 것일 수 있다. 구체적으로, 상기 이형 필름은 상기 다공성 생분해성 고분자 필름의 이면에서 최외곽층으로 포함되는 것일 수 있다. 이때 사용되는 이형 필름으로는 불소가 코팅된 폴리에틸렌 테레프탈레이트(polyethylene terephthalate; PET) 필름을 사용할 수 있으나, 이에 한정되는 것은 아니다.A release film may be attached to the back surface of the porous biodegradable polymer film. Specifically, the release film may be included as the outermost layer on the back side of the porous biodegradable polymer film. As the release film used at this time, a fluorine-coated polyethylene terephthalate (PET) film may be used, but is not limited thereto.
상기 다공성 생분해성 고분자 필름은 폴리락트산(Poly(lactic acid)), 폴리글리콜산(Poly(glycolic acid)), 폴리락틱글리콜산(Poly(latic-co-glycolic acid)), 폴리디메틸실록산((Poly)dimethylsiloxane), 셀룰로오스(Cellulose), 폴리하이드록시뷰틸레이트 하이드록시발레레이트(Poly(hydroxybutyrate hydroxyvalerate)), 폴리에틸렌글리콜(Polyethylene glycol) 및/또는 폴리카프로락톤(Poly-ε-caprolactone) 필름일 수 있으나, 이에 제한되는 것은 아니다.The porous biodegradable polymer film is polylactic acid (Poly (lactic acid)), polyglycolic acid (Poly (glycolic acid)), polylactic-glycolic acid (Poly (latic-co-glycolic acid)), polydimethylsiloxane ((Poly )dimethylsiloxane), cellulose, polyhydroxybutyrate hydroxyvalerate (Poly (hydroxybutyrate hydroxyvalerate)), polyethylene glycol and / or poly-ε-caprolactone (Poly-ε-caprolactone) film, It is not limited thereto.
상기 고분자 필름은 고분자 분자구조 내에 아마이드 결합, 에스터 결합 또는 글리코시딕 결합을 갖고 있어 생분해 가능하며, 짧게는 몇 주에서 길게는 몇 달 이내에 자연에서 분해되기 때문에 자연 친화적인 장점을 가지고 있다.The polymer film has an amide bond, an ester bond, or a glycosidic bond in the molecular structure of the polymer, so it is biodegradable, and it has a nature-friendly advantage because it is degraded in nature within a few weeks to a few months as short as possible.
본 발명의 또 다른 일 구현예에 따르면, 본 발명은 태반-유래 줄기세포가 배양되어 있는 다공성 생분해성 고분자 필름 이면에 생체적합성 고분자 지지체(하이드로젤)가 부착되어 있는 바이오패치형 세포치료제에 관한 것이다.According to another embodiment of the present invention, the present invention relates to a biopatch-type cell therapy agent in which a biocompatible polymer scaffold (hydrogel) is attached to the back of a porous biodegradable polymer film in which placenta-derived stem cells are cultured.
상기 생체적합성 고분자 지지체는 세포 배양을 위한 영양성분을 함유하는 것일 수 있다. 구체적으로, 세포 배양을 위한 영양성분은 세포외기질(extracellular matrix)을 포함하는 것일 수 있으나, 이에 제한되는 것은 아니다.The biocompatible polymer scaffold may contain nutrients for cell culture. Specifically, nutrients for cell culture may include, but are not limited to, an extracellular matrix.
상기 생체적합성 고분자 지지체는 태반-유래 줄기세포가 배양되어 있는 다공성 생분해성 고분자 필름 이면에 부착되어, 다공성의 생분해성 고분자 필름의 구멍을 통해 세포 배양을 위한 영양성분(줄기세포 배양액 포함)을 세포에 공급함으로써, 세포의 활성을 유지하고 환부의 재생(치료) 효과를 극대화하는 기능을 할 수 있다. 추가적으로, 환부로부터의 삼출물을 담아내는 역할도 부수적으로 할 수 있다.The biocompatible polymer scaffold is attached to the back of the porous biodegradable polymer film in which placenta-derived stem cells are cultured, and nutrients for cell culture (including stem cell culture medium) are supplied to the cells through pores of the porous biodegradable polymer film. By supplying it, it can function to maintain cell activity and maximize the effect of regeneration (treatment) on the affected area. In addition, the role of containing the exudate from the affected area can also be performed incidentally.
상기 생체적합성 고분자 지지체는 생체적합성 고분자로 이루어진 일정 두께를 갖는 얇은 막 형태의 구조물을 말하며, 원하는 형태로 잘라서 사용할 수 있는 장점이 있다.The biocompatible polymer scaffold refers to a structure in the form of a thin film having a certain thickness made of a biocompatible polymer, and has the advantage of being cut into a desired shape for use.
상기 생체적합성 고분자 지지체는 0.05 내지 10.0 ㎜의 두께를 가질 수 있으나, 이에 제한되는 것은 아니다.The biocompatible polymer scaffold may have a thickness of 0.05 to 10.0 mm, but is not limited thereto.
상기 생체적합성 고분자 지지체는 젤라틴, 히알루론산, 헤파린, 셀룰로스, 덱스트란, 알지네이트, 키토산, 키틴, 콜라겐, 콘드로이틴황산 및/또는 펙틴 지지체일 수 있으나, 이에 제한되는 것은 아니다.The biocompatible polymer scaffold may be gelatin, hyaluronic acid, heparin, cellulose, dextran, alginate, chitosan, chitin, collagen, chondroitin sulfate, and/or pectin scaffold, but is not limited thereto.
본 발명의 바람직한 구현예에 따르면, 본 발명의 바이오패치형 세포치료제는 태반-유래 줄기세포가 배양되어 있고 다공성을 가지는 생분해성 고분자 필름에 생체적합성 고분자 지지체가 적층된 형태를 가질 수 있다.According to a preferred embodiment of the present invention, the biopatch-type cell therapy product of the present invention may have a form in which placenta-derived stem cells are cultured and a biocompatible polymer scaffold is laminated on a porous biodegradable polymer film.
도 14는 본 발명의 일 실시예에 따른 바이오패치형 세포치료제의 적층 구조를 나타낸 것이다.14 shows a laminated structure of a biopatch-type cell therapy product according to an embodiment of the present invention.
도 14를 참조하면, 본 발명의 바이오패치형 세포치료제는 태반-유래 줄기세포가 배양된 다공성 생분해성 필름과 세포 배양을 위한 영양성분이 함유된 생체적합성 고분자 지지체(하이드로젤)를 포함하는 형태로 구성될 수 있다.Referring to FIG. 14, the biopatch-type cell therapy product of the present invention is composed of a porous biodegradable film in which placenta-derived stem cells are cultured and a biocompatible polymer scaffold (hydrogel) containing nutrients for cell culture. It can be.
본 발명의 바람직한 구현예에 따르면, 상기와 같은 본 발명에 따른 바이오패치형 세포치료제를 이용하는 하나의 실시예로서, 본 발명의 바이오패치형 세포치료제를 이용하는 피부 재생 방식을 설명하면 다음과 같다.According to a preferred embodiment of the present invention, as an example of using the biopatch-type cell therapy agent according to the present invention as described above, a skin regeneration method using the biopatch-type cell therapy agent of the present invention will be described as follows.
도 14에서와 같이, 태반-유래 줄기세포가 배양된 다공성 생분해성 고분자 필름(A) 및 생체적합성 고분자 지지체(하이드로젤)(B)가 적층된 바이오패치를 적당한 크기로 절단하거나 절단하지 아니하고 적용 부위에 붙인다. 여기서 적용 부위로는 예컨대 화상환자 치료 부위 등 다양한 적용 부위가 고려될 수 있다.As shown in FIG. 14, the biopatch in which the porous biodegradable polymer film in which placenta-derived stem cells are cultured (A) and the biocompatible polymer scaffold (hydrogel) (B) are laminated is cut into an appropriate size or applied to the site without cutting. stick to Here, as the application site, various application sites such as, for example, a burn patient treatment site may be considered.
본 발명의 바람직한 구현예에 따르면, 적용 부위에 붙이는 과정에서는 생체적합성 고분자 지지체에 추가적으로 점착제(C)를 덧대어 부착함으로써, 적용 부위(피부)에 부착하는 형태로 적용될 수 있다.According to a preferred embodiment of the present invention, in the process of attaching to the application site, it can be applied in the form of attachment to the application site (skin) by additionally attaching the adhesive (C) to the biocompatible polymer support.
본 발명의 바람직한 구현예에 따르면, 이와 같은 방법으로 바이오패치형 세포치료제를 적용하면, 다공성 생분해성 고분자 필름 표면에 배양된 줄기세포가 천공된 구멍을 따라 아래로 이동하고 피부 표면에 생착하여 성장하면서 매우 효과적인 피부 조직 재생 활성을 발휘할 수 있다. 상당한 시간 경과 후, 다공성 생분해성 고분자 필름은 분해되어 없어지고 활착 된 세포로 인한 재생 효과를 발현하게 되는 것이다.According to a preferred embodiment of the present invention, when the biopatch-type cell therapy agent is applied in this way, the stem cells cultured on the surface of the porous biodegradable polymer film move downward along the perforated hole, engraft to the skin surface, and grow while growing. It can exert an effective skin tissue regeneration activity. After a considerable period of time, the porous biodegradable polymer film decomposes and disappears, and the regenerative effect due to the established cells is expressed.
본 발명은 태반-유래 줄기세포를 포함하는 피부 재생용 바이오패치형 세포치료제에 관한 것으로, 생분해성 고분자 필름에 태반-유래 줄기세포가 존재하는 상태 그대로 피부 등 생체 이식이 가능하기 때문에 오염이나 조작의 복잡함이 없이 간편하게 생체 이식 소재로 활용할 수 있는 효과가 있다.The present invention relates to a biopatch-type cell therapy product for skin regeneration containing placenta-derived stem cells, which can be transplanted into a living body such as the skin in a state where placenta-derived stem cells are present in a biodegradable polymer film, so contamination or manipulation is complicated. There is an effect that it can be used as a living body transplant material easily without this.
도 1은 말의 양막 조직을 멸균된 배양 배지에 옮긴 모습이다.Figure 1 is a state of transfer of equine amnion tissue to a sterile culture medium.
도 2는 본 발명의 일 실시예에 따라, 본 발명의 양막 유래 세포의 초기 배양 결과를 나타낸다.Figure 2 shows the results of initial culture of the amnion-derived cells of the present invention according to one embodiment of the present invention.
도 3은 본 발명의 일 실시예에 따라, 본 발명의 양막 유래 줄기세포의 자가 복제 및 증식 능력을 확인한 것으로, 도 3A는 초기 계대부터 45 계대 이상의 양막 유래 줄기세포의 Doubling time, 도 3B는 양막 유래 줄기세포와 피부 유래 섬유아세포의 doubling time을 비교한 그래프이다. Figure 3 confirms the ability of self-replication and proliferation of the amniotic membrane-derived stem cells of the present invention according to an embodiment of the present invention. This is a graph comparing the doubling time of stem cells and skin-derived fibroblasts.
도 4는 양막 유래 줄기세포와 피부 유래 섬유아세포의 콜로니 형성 능력을 비교한 결과이다.4 is a result of comparing the colony formation abilities of amnion-derived stem cells and skin-derived fibroblasts.
도 5는 본 발명의 일 실시예에 따라, 본 발명의 양막 유래 줄기세포의 지방세포로의 분화 능력을 확인한 결과이다. (A: 분화 유도 전(X100), B: 배양 7일 후(X100), C: 배양 7일 후(X200))5 is a result of confirming the differentiation ability of amniotic membrane-derived stem cells of the present invention into adipocytes according to an embodiment of the present invention. (A: before differentiation induction (X100), B: after 7 days of culture (X100), C: after 7 days of culture (X200))
도 6은 본 발명의 일 실시예에 따라, 본 발명의 양막 유래 줄기세포의 골아세포로의 분화 능력을 확인한 결과이다. (A: 분화 유도 전(X100), B: 배양 7일 후(X100), C: 배양 7일 후(X200))6 is a result of confirming the differentiation ability of the amniotic membrane-derived stem cells of the present invention into osteoblasts according to an embodiment of the present invention. (A: before differentiation induction (X100), B: after 7 days of culture (X100), C: after 7 days of culture (X200))
도 7은 본 발명의 일 실시예에 따라, 본 발명의 양막 유래 줄기세포의 연골세포로의 분화 능력을 확인한 결과이다. (A: 분화 유도 전(X100), B: 배양 7일 후(X100), C: 배양 7일 후(X200))7 is a result of confirming the differentiation ability of amniotic membrane-derived stem cells of the present invention into chondrocytes according to an embodiment of the present invention. (A: before differentiation induction (X100), B: after 7 days of culture (X100), C: after 7 days of culture (X200))
도 8은 본 발명의 일 실시예에 따라, 역전사 중합효소 연쇄반응(RT-PCR)을 이용하여 본 발명의 양막 유래 줄기세포의 다능성을 확인한 결과이다. (컨트롤 마커: beta-actin)8 is a result of confirming the pluripotency of amniotic membrane-derived stem cells of the present invention using reverse transcription polymerase chain reaction (RT-PCR) according to an embodiment of the present invention. (Control marker: beta-actin)
도 9는 본 발명의 일 실시예에 따라, 실시간 정량(qRT-PCR)을 이용하여 본 발명의 양막 유래 줄기세포의 다능성을 확인한 결과이다. 9 is a result of confirming the pluripotency of the amniotic membrane-derived stem cells of the present invention using real-time quantitative (qRT-PCR) according to an embodiment of the present invention.
도 10은 본 발명의 일 실시예에 따라, 유세포 분석을 이용하여 본 발명의 양막 유래 줄기세포의 다능성을 확인한 결과이다. (red line: negative control, blue line: 양성 발현 데이터)10 is a result of confirming the pluripotency of the amniotic membrane-derived stem cells of the present invention using flow cytometry according to an embodiment of the present invention. (red line: negative control, blue line: positive expression data)
도 11은 본 발명의 일 실시예에 따라 제작된 다공성 필름 세포 배양면의 형태를 나타낸다. 11 shows the shape of a porous film cell culture surface manufactured according to an embodiment of the present invention.
도 12는 본 발명의 일 실시예에 따라 제작된 다공성 필름 세포 배양면에 대하여, 플라즈마 처리 조건별 PLLA/SSA 필름의 세포 생착률을 확인한 것으로, 도 12A는 인간 혈관내피 유래 암세포(Human hepatic adenocarcinoma cell line, sk-hep1 cells), 도 12B는 인간 피부 유래 섬유아세포(Human skin Fibroblast, CCD-986sk)에 대한 결과이다.Figure 12 confirms the cell engraftment rate of the PLLA/SSA film for each plasma treatment condition with respect to the porous film cell culture surface manufactured according to an embodiment of the present invention. Figure 12A is human hepatic adenocarcinoma cell line , sk-hep1 cells), and FIG. 12B is a result of human skin fibroblast (CCD-986sk).
도 13은 본 발명의 일 실시예에 따른 하이드로젤 제작용 실리콘 몰드의 모식도를 나타낸다. 몰드를 통해 제작된 하이드로젤(C 및 F)은 젤라틴과 동결건조한 중간엽 줄기세포 배양액을 함유한다.13 shows a schematic diagram of a silicone mold for producing a hydrogel according to an embodiment of the present invention. The hydrogels (C and F) fabricated through the mold contain gelatin and lyophilized mesenchymal stem cell culture medium.
도 14는 본 발명의 일 실시예에 따른 바이오패치형 세포치료제의 모식도를 나타낸다. 줄기세포가 배양된 필름(A), 하이드로젤(B), 점착제(Backing layer, C)로 구성된다. 14 shows a schematic diagram of a biopatch-type cell therapy product according to an embodiment of the present invention. It is composed of stem cell-cultured film (A), hydrogel (B), and adhesive (Backing layer, C).
도 15는 본 발명의 일 실시예에 따라, 본 발명의 바이오패치형 세포치료제의 유무에 따른 세포 형태를 확인한 결과이다. (A: 바이오패치가 없는 경우, B: 바이오패치에 세포를 배양한 경우)15 is a result of confirming the cell morphology according to the presence or absence of the biopatch-type cell therapy agent of the present invention according to an embodiment of the present invention. (A: In case of no biopatch, B: In case of culturing cells in biopatch)
도 16은 본 발명의 일 실시예에 따라, 본 발명의 바이오패치형 세포치료제의 유무에 따른 줄기세포 증식 능력을 확인한 결과이다. 16 is a result of confirming stem cell proliferation ability according to the presence or absence of the biopatch-type cell therapy agent of the present invention according to an embodiment of the present invention.
도 17은 본 발명의 일 실시예에 따라, 본 발명의 바이오패치형 세포치료제의 유무에 따른 세포 활성 및 생존 능력을 확인한 결과이다.17 is a result of confirming cell activity and viability according to the presence or absence of the biopatch-type cell therapy agent of the present invention according to an embodiment of the present invention.
도 18은 본 발명의 일 실시예에 따라, 본 발명의 바이오패치형 줄기세포 치료제의 피부 치료 효능 유효성을 평가를 위하여, 바이오패치형 줄기세포 치료제 이식 과정을 나타낸 도이다.18 is a diagram showing a biopatch-type stem cell treatment implantation process in order to evaluate the skin treatment efficacy and effectiveness of the biopatch-type stem cell treatment according to an embodiment of the present invention.
도 19는 본 발명의 일 실시예에 따라, 본 발명의 바이오패치형 줄기세포 치료제가 이식된 피부 손상 부위에 대한 시간 경과에 따른 상처 면적 변화를 확인한 것으로, 도 19A는 상처 면적을 측정한 사진, 도 19B는 상처 면적 대비 시간 경과 후의 상처 면적을 %로 도출하여 나타낸 결과이다.Figure 19 is a confirmation of the change in the wound area over time for the damaged skin area to which the biopatch-type stem cell therapeutic agent of the present invention has been transplanted according to an embodiment of the present invention, and Figure 19A is a photograph of measuring the wound area; 19B is the result obtained by deriving the wound area after the passage of time as a percentage compared to the wound area.
도 20은 본 발명의 일 실시예에 따라, 본 발명의 바이오패치형 줄기세포 치료제가 이식된 피부 손상 부위에 대한 피부 재생 효과를 확인한 것으로, 도 20A는 손상된 피부의 H&E 조직 염색을 통한 조직학적 분석 결과, 도 20B는 조직 염색 사진에서 정상 피부 조직 끝부분(Skin margin)에서부터 재상피화가 진행된 부분까지의 길이를 수치화 하여 나타낸 결과이다.Figure 20 confirms the skin regeneration effect on the damaged skin area to which the biopatch-type stem cell therapeutic agent of the present invention has been transplanted according to an embodiment of the present invention, and Figure 20A is the result of histological analysis through H&E tissue staining of the damaged skin , Figure 20B is a result of digitizing the length from the normal skin tissue end (Skin margin) to the re-epithelialized part in the tissue staining photograph.
본 발명은 태반-유래 줄기세포(Placenta-derived stem cells)로, 다공성 생분해성 고분자 필름 및 생체적합성 고분자 지지체를 포함하는, 바이오패치형 세포치료제에 관한 것이다.The present invention relates to a biopatch-type cell therapy product comprising placenta-derived stem cells, a porous biodegradable polymer film, and a biocompatible polymer scaffold.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
실시예 1. 태반 유래 세포의 분리 및 배양Example 1. Isolation and culture of placenta-derived cells
임신이 확인된 암말 분만 시, 모체와 분리되어 새끼를 감싸고 있는 양막 조직을 멸균된 수술 도구를 사용하여 멸균된 통으로 옮긴 후, 약 4 ℃를 유지한 채로 평균 약 3시간 내에 실험실로 운반하였다. 운반된 양막 조직을 UV 살균한 무균 작업대 안에서 70% 에탄올에 담가 1회 소독한 뒤, 5% 페니실린/스트렙토마이신을 포함하는 PBS(phosphate Buffered Saline)로 3회 이상 세척하였다. 세척된 양막 조직을 멸균 접시로 옮긴 후(도 1 참조), 멸균된 수술용 메스를 사용하여 잘게 다진 다음 멸균 튜브로 옮기고 2500rpm, 7분 동안 원심 분리하여 펠렛(pellet)을 회수하였다. 회수한 펠렛으로부터 상기 세척(5% 페니실린/스트렙토마이신을 포함하는 PBS) 및 원심 분리(2500rpm, 7분) 과정을 2회 진행하여 단핵세포를 분리하였다.At the time of delivery of a mare whose pregnancy was confirmed, the amnion tissue that was separated from the mother and surrounding the foal was transferred to a sterile tub using a sterilized surgical tool, and then transported to the laboratory within an average of about 3 hours while maintaining about 4 ° C. The transported amniotic tissue was sterilized once by soaking in 70% ethanol in a UV sterilized aseptic workbench, and then washed three or more times with PBS (phosphate buffered saline) containing 5% penicillin/streptomycin. The washed amnion tissue was transferred to a sterile dish (see FIG. 1), minced using a sterilized scalpel, transferred to a sterilized tube, and centrifuged at 2500 rpm for 7 minutes to recover a pellet. Mononuclear cells were isolated from the recovered pellet by performing the washing (PBS containing 5% penicillin/streptomycin) and centrifugation (2500 rpm, 7 minutes) process twice.
분리된 양막 유래 단핵세포의 초기 배양을 위하여, 15% FBS(fetal bovine serum), 1% Glutamax 및 1% 페니실린/스트렙토마이신이 포함된 DMEM(dulbecco Modified Eagle Medium) 배양 배지를 사용하였다. 이때, 세포 부착성을 높이기 위해 배양 접시를 0.1% 젤라틴으로 코팅하여 배양하였으며, 말과(Equidae) 동물의 체온과 같은 38.5 ℃에서 5% CO2 환경의 인큐베이터에 넣고 배양하였다.For the initial culture of isolated amniotic membrane-derived mononuclear cells, DMEM (dulbecco Modified Eagle Medium) culture medium containing 15% fetal bovine serum (FBS), 1% Glutamax, and 1% penicillin/streptomycin was used. At this time, in order to increase cell adhesion, the culture dish was coated with 0.1% gelatin and cultured, and the equine ( Equidae ) was placed in an incubator in a 5% CO 2 environment at 38.5 ° C., the same as the body temperature of an animal, and cultured.
죽은 조직 및 세포를 제거해주기 위해 초기 배양 시작 48h 안에 배양 배지를 교체하였고, 세포가 배양 접시 바닥에 부착된 것이 관찰되었을 경우에도 배양 배지를 교체하였으며, 배양 배지 바닥의 80% 이상 세포가 부착되면 계대 또는 동결하여 보관하였다. To remove dead tissues and cells, the culture medium was replaced within 48 h of the initial culture, and the culture medium was also replaced when cells were observed to adhere to the bottom of the culture dish, and passage was performed when more than 80% of the cells attached to the bottom of the culture medium or frozen and stored.
그 결과, 양막 유래 세포들은 평균 약 5 일 후 부착된 형태로 관찰할 수 있었으며, 도 2에서 확인할 수 있듯이, 세포의 형상은 섬유아세포와 유사한 입방(spindle)형으로 관찰되었으며(도 2의 A), 이는 다른 중간엽 줄기세포에서 나타나는 형상과 유사하다. 추가적으로, 세포 형태의 자세한 관찰(X100)을 위하여 Diff-quik으로 염색하여 확인하였다(도 2의 B).As a result, the amnion-derived cells could be observed in an attached form after an average of about 5 days, and as can be seen in FIG. 2, the shape of the cells was observed in a spindle shape similar to that of fibroblasts (FIG. 2A). , which is similar to that seen in other mesenchymal stem cells. Additionally, for detailed observation of cell morphology (X100), it was confirmed by staining with Diff-quik (FIG. 2B).
실시예 2. 태반 유래 줄기세포의 자가 복제 및 증식 능력 확인(doubling time analysis)Example 2. Confirmation of self-replication and proliferation ability of placenta-derived stem cells (doubling time analysis)
상기 실시예 1에서 보관된 양막 유래 줄기세포를 6-웰 배양 접시에 200,000 cell/well의 밀도로 분주한 후 새로운 배지를 전혀 첨가하지 않는 방법으로 38.5 ℃, 5% CO2의 인큐베이터에 넣고 배양하여 배양 시간 및 전체 활성 세포수를 관측하였다. 대조군으로 피부 유래 섬유아세포를 사용하였다.The amniotic membrane-derived stem cells stored in Example 1 were dispensed at a density of 200,000 cells/well in a 6-well culture dish, and then placed in an incubator at 38.5 °C, 5% CO 2 and cultured without adding any new medium. Incubation time and total number of active cells were observed. As a control, skin-derived fibroblasts were used.
Doubling time 분석은 세포가 처음의 수에서 두 배가 되는 시간 분석을 통해 세포의 증식율을 나타내는 지수로 표현되며, 다음과 같은 계산식으로 표현된다. Doubling time analysis is expressed as an index representing the cell proliferation rate through the analysis of the time at which cells double from the initial number, and is expressed in the following formula.
[계산식][formula]
Doubling time(hr) ={(T-Tc)log2 }/(logN - logNc)Doubling time(hr) ={(T-Tc)log2 }/(logN - logNc)
T-T0: 세포 배양 시간(hr)TT 0 : cell culture time (hr)
N0: 초기 분주한 세포 수, N: 최종 배양된 세포 수N 0 : number of cells initially seeded, N: number of final cultured cells
활성 세포(viable cell)는 트리판 블루(trypan blue) 염색을 이용하여 계측하였다. 구체적으로, 6-웰 배양 접시 내의 배지를 제거하고 PBS로 세척 후 0.05% Typsin-EDTA를 이용하여 세포를 기저면에서 분리시켰다. 이후 새로운 5% FBS가 포함된 배양액으로 Trypsin의 작용을 중지시키고, 세포 배양 접시에서 분리된 세포를 계측하였다.Viable cells were counted using trypan blue staining. Specifically, the medium in the 6-well culture dish was removed, washed with PBS, and cells were detached from the basal surface using 0.05% Typsin-EDTA. Then, the action of Trypsin was stopped with a culture medium containing 5% FBS, and the cells separated from the cell culture dish were counted.
그 결과, 도 3a 및 3b에서 확인할 수 있듯이, 치료 능력이 없는 피부 유래 섬유아세포(Skin cell)에 비하여 본 발명의 양막 유래 줄기세포(AM-MSC)의 doubling time이 낮게 나타났다. 이는, 배양된 세포가 분열하여 처음 배양했던 수의 두 배가 되는 시점이 더 빠르다는 것으로, 본 발명의 양막 유래 줄기세포의 우수한 자가 복제 및 증식 능력을 시사한다.As a result, as can be seen in Figures 3a and 3b, the doubling time of the amniotic membrane-derived stem cells (AM-MSC) of the present invention was lower than that of skin-derived fibroblasts (Skin cells) having no therapeutic ability. This indicates that the time point at which the cultured cells divide and double the number initially cultured is faster, suggesting the excellent self-replication and proliferation ability of the amniotic membrane-derived stem cells of the present invention.
또한 도 4에서 확인할 수 있듯이, 치료 능력이 없는 피부 유래 섬유아세포(Skin fibroblast)에 비하여 본 발명의 양막 유래 줄기세포(Equine AM)의 콜로니 숫자 및 크기가 월등하게 나타났다. 이는, 본 발명의 양막 유래 줄기세포의 우수한 자가 재생 능력을 시사한다.In addition, as can be seen in Figure 4, the colony number and size of the amnion-derived stem cells (Equine AM) of the present invention were superior to those of skin-derived fibroblasts having no therapeutic ability. This suggests the excellent self-renewal ability of the amniotic membrane-derived stem cells of the present invention.
실시예 3. 태반 유래 줄기세포의 분화 능력 확인Example 3. Confirmation of differentiation ability of placenta-derived stem cells
양막 유래 중간엽 줄기세포의 분화 능력을 확인하기 위하여, StemPro Differentiation Kit(Gibco, USA)를 사용하여 각각 계통에 맞는 배지에서 지방세포, 연골세포, 골세포로 분화를 유도하였다.In order to confirm the differentiation ability of amniotic membrane-derived mesenchymal stem cells, differentiation into adipocytes, chondrocytes, and osteocytes was induced in a medium suitable for each lineage using the StemPro Differentiation Kit (Gibco, USA).
1) 지방세포 분화(Adipogenic differentiation)1) Adipogenic differentiation
StemPro Adipogenesis Differentiation Kit를 이용하여 6-웰 배양 접시에 200,000 cell/well의 밀도로 분주하고, 38.5 ℃, 5 % CO2의 인큐베이터에 넣고 배양하였다. 2일에 한 번씩 배지를 교환하였다. 오일 레드 오(Oil Red O)로 염색하여 분화 여부를 확인하였다.It was dispensed at a density of 200,000 cell/well in a 6-well culture dish using the StemPro Adipogenesis Differentiation Kit, and cultured into an incubator at 38.5 °C and 5% CO 2 . Medium was exchanged once every two days. Differentiation was confirmed by staining with Oil Red O.
그 결과, 도 5에서 확인할 수 있듯이, 분화 유도 전에는 양막 줄기세포의 염색 음성 반응이 나타났으나(X100, 도 5의 A), 배양 약 7일 후 양막 유래 중간엽 줄기세포 모두 섬유아세포 형태에서 둥근 모양으로 세포의 형태가 변화한 것을 알 수 있었으며, 지질 방울 형성으로 인한 염색을 관찰할 수 있었다(X100, 도 5의 B; X200, 도 5의 C).As a result, as can be seen in FIG. 5, before the induction of differentiation, staining-negative reactions of amnion stem cells were observed (X100, FIG. 5A), but after about 7 days of culture, all amnion-derived mesenchymal stem cells were round in the form of fibroblasts. It was found that the shape of the cells changed in shape, and staining due to the formation of lipid droplets was observed (X100, FIG. 5B; X200, FIG. 5C).
2) 골아세포 분화(Osteogenic differentiation)2) Osteogenic differentiation
StemPro Osteogenesis Differentiation Kit를 이용하여 6-웰 배양 접시에 200,000 cell/well의 밀도로 분주하고, 38.5 ℃, 5 % CO2의 인큐베이터에 넣고 배양하였다. 2일에 한 번씩 배지를 교환하였다. 알리자린 레드 에스(alizarin red S)로 염색하여 분화 여부를 확인하였다.It was dispensed at a density of 200,000 cell/well in a 6-well culture dish using the StemPro Osteogenesis Differentiation Kit, and cultured in an incubator at 38.5 °C and 5% CO 2 . Medium was exchanged once every two days. Differentiation was confirmed by staining with alizarin red S.
그 결과, 도 6에서 확인할 수 있듯이, 분화 유도 전에는 양막 줄기세포의 염색 음성 반응이 나타났으나(X100, 도 6의 A), 배양 약 7일 후 양막 유래 중간엽 줄기세포 모두 섬유아세포 형태에서 입방형으로 세포의 형태가 변화한 것을 알 수 있었으며, 배양 약 21일 후 세포 외 기질(extracellular matrix)에 칼슘(calcium) 침전으로 인한 염색을 관찰할 수 있었다(X100, 도 6의 B; X200, 도 6의 C).As a result, as can be seen in FIG. 6, before the induction of differentiation, staining-negative reactions of amnion stem cells were observed (X100, FIG. 6A), but after about 7 days of culture, all amnion-derived mesenchymal stem cells were cubic in the form of fibroblasts. It was found that the morphology of the cells changed to the same type, and after about 21 days of culture, staining due to calcium precipitation in the extracellular matrix was observed (X100, Fig. 6B; X200, Fig. 6). 6 C).
3) 연골세포 분화(Chondrogenic differentiation)3) Chondrogenic differentiation
StemPro Chondrogenesis Differentiation Kit를 이용하여 1,000 cells/μL의 농도로 행잉 드롭(hanging drop)하고, 세포가 구 형태로 자라도록 38.5 ℃, 5 % CO2의 인큐베이터에 넣고 배양하였다. 3일에 한 번씩 배지를 추가 또는 교환하였다. 알시안 블루(alcian blue)로 염색하여 분화 여부를 확인하였다.Using the StemPro Chondrogenesis Differentiation Kit, a hanging drop was performed at a concentration of 1,000 cells/μL, and cells were placed in an incubator at 38.5° C. and 5% CO 2 to grow in a spherical shape and cultured. Medium was added or exchanged every 3 days. Differentiation was confirmed by staining with alcian blue.
그 결과, 도 7에서 확인할 수 있듯이, 분화 유도 전에는 양막 줄기세포의 염색 음성 반응이 나타났으나(X100, 도 7의 A), 배양 약 14 일 후 연골세포 분화로 인한 염색을 관찰할 수 있었다(X100, 도 7의 B; X200, 도 7의 C).As a result, as can be seen in FIG. 7 , staining of amnion stem cells was negative before induction of differentiation (X100, FIG. 7A), but after about 14 days of culture, staining due to chondrocyte differentiation was observed ( X100, Fig. 7B; X200, Fig. 7C).
실시예 4. 태반 유래 줄기세포의 다능성 확인Example 4. Verification of pluripotency of placenta-derived stem cells
국제 줄기세포 위원회(International society for cellular therapy, ISCT)에서 제시한 말과(Equidae) 동물의 중간엽 줄기세포에서 양성 발현을 보이는 CD44, CD90, CD105, 음성 발현을 보이는 CD34, CD38, CD45 및 면역 관련 마커 MHC class II에 대한 분석을 수행하였다. 또한, 양막 유래 줄기세포의 특성으로 알려진 배아줄기세포 관련 유전자 Oct4, c-Myc, Klf4 및 중간엽 줄기세포 관련 유전자 PAX6, ALCAM, Integrin-β1, Endoglin, HCAM에 대하여 mRNA 레벨에서의 발현 양상을 분석하였다.CD44, CD90, CD105 showing positive expression in mesenchymal stem cells of Equidae animals presented by the International Society for Cellular Therapy (ISCT), CD34, CD38, CD45 showing negative expression, and immune-related Analysis for the marker MHC class II was performed. In addition, the expression patterns at the mRNA level were analyzed for embryonic stem cell related genes Oct4, c-Myc, Klf4 and mesenchymal stem cell related genes PAX6, ALCAM, Integrin-β1, Endoglin, and HCAM, known as characteristics of amniotic membrane-derived stem cells. did
1) 역전사 중합효소 연쇄반응(Reverse transcriptase polymerase chain reaction, RT-PCR)1) Reverse transcriptase polymerase chain reaction (RT-PCR)
Total RNA는 제조사의 지침에 따라 RNA 추출 키트를 사용하여 준비하였다 (Qiagen, Cat. No. 74104). RNA 시료(Total RNA 1㎕)는 iScript 역전사효소(BIO-RAD)를 사용하여 올리고 dT 프라이머 및 랜덤(random) 프라이머로 cDNA를 합성하였다. 구체적인 프라이머 서열은 하기 표 1에 나타내었다.Total RNA was prepared using an RNA extraction kit according to the manufacturer's instructions (Qiagen, Cat. No. 74104). For RNA samples (1 μl of total RNA), cDNA was synthesized with oligo dT primers and random primers using iScript reverse transcriptase (BIO-RAD). Specific primer sequences are shown in Table 1 below.
RT-PCR은 95 ℃에서 4분간 변성 후, 95 ℃에서 30초, 56 ℃에서 30초, 및 72 ℃에서 30초를 34 회 반응시켰다. 73 ℃에서 5분간 최종 연장하였다. 베타 액틴(beta-Actin) RT-PCR 반응은 34 회 및 60 ℃에서의 프라이머 어닐링(annealing)으로 변형하여 수행하였다. 표적 유전자의 RT-PCR 특이적 증폭은 DNA 염기서열 분석으로 확인하였다.그 결과, 도 8에서 확인할 수 있듯이, 배아줄기세포 관련 유전자(Oct4, c-Myc, 및 Klf4) 및 중간엽 줄기세포 관련 유전자(PAX6, ALCAM, Integrin-beta1, Endoglin, 및 HCAM) 모두 발현되는 것을 알 수 있었다.2) 실시간 정량(quantitative real time PCR, qRT-PCR) RT-PCR was denatured at 95 °C for 4 minutes, then reacted 34 times at 95 °C for 30 seconds, 56 °C for 30 seconds, and 72 °C for 30 seconds. A final extension was performed at 73 °C for 5 minutes. Beta-Actin RT-PCR reaction was performed by modifying the primer annealing at 34 times and 60 °C. RT-PCR specific amplification of the target gene was confirmed by DNA sequencing. As a result, as shown in FIG. 8, embryonic stem cell-related genes (Oct4, c-Myc, and Klf4) and mesenchymal stem cell-related genes (PAX6, ALCAM, Integrin-beta1, Endoglin, and HCAM) were all expressed. 2) Quantitative real time PCR (qRT-PCR)
상기 RT-PCR에 사용했던 cDNA 합성 방법과 동일한 방법으로 cDNA를 합성하였다. qRT-PCR은 95 ℃에서 10분간 변성 후, 94 ℃에서 15초, 58 ℃에서 30초, 및 72 ℃에서 30초를 39 회 반응시켰다. 95 ℃에서 10초, 65 ℃에서 5초 간 반응시킨 후 대조군인 말의 피부 유래 섬유아세포와 마커 발현양을 비교하였다. cDNA was synthesized by the same method as the cDNA synthesis method used for the RT-PCR. qRT-PCR was denatured at 95 °C for 10 minutes, followed by 39 reactions at 94 °C for 15 seconds, 58 °C for 30 seconds, and 72 °C for 30 seconds. After reacting at 95 ℃ for 10 seconds and 65 ℃ for 5 seconds, the amount of marker expression was compared with that of control horse skin-derived fibroblasts.
그 결과, 도 9에서 확인할 수 있듯이, 치료 능력이 없는 피부 유래 섬유아세포(Skin)에 비하여 본 발명의 양막 유래 줄기세포(AM-MSC)에서 다능성을 나타내는 유전자(Oct4, c-Myc, 및 Klf4) 및 줄기세포에 특이적으로 나타나는 유전자(PAX6, ALCAM, Integrin-beta1, Endoglin, 및 HCAM) 모두 유의적으로 높게 발현되는 것을 알 수 있었다.As a result, as can be seen in FIG. 9, genes (Oct4, c-Myc, and Klf4) exhibiting pluripotency in amnion-derived stem cells (AM-MSC) of the present invention compared to skin-derived fibroblasts (Skin) having no therapeutic ability. ) and stem cell-specific genes (PAX6, ALCAM, Integrin-beta1, Endoglin, and HCAM) were all significantly highly expressed.
3) 유세포 분석(Flow cytometry analysis)3) Flow cytometry analysis
FITC(fluorescein isothiocyanate) 또는 PE(phycoerythrin)와 결합된 다양한 조합의 항체 또는 순수한 1차 항체를 양막 유래 줄기세포에 붙인 뒤 형광이 붙어있는 2차 IgG 항체를 사용하여 유세포 분석을 통해 세포 표지 인자를 분석하였다. 2차 IgG 항체를 기준으로 상대적인 발현 양상 비교를 위해 사용된 항체는 다음과 같다: CD29-PE, CD34-FITC, CD38-FITC, CD44-FITC, CD45, CD90-PE, CD105-FITC, MHC class II-FITC 및 IgG-FITC. 시료 당 약 5~8x105 개의 세포가 하나의 항체를 분석하는 데 사용되었으며, Cellquest 소프트웨어를 사용하여 유세포 분석(FACScan, BD Biosciences, USA)을 실시하였다.Various combinations of antibodies or pure primary antibodies coupled with FITC (fluorescein isothiocyanate) or PE (phycoerythrin) are attached to amnion-derived stem cells, and then the cell markers are analyzed by flow cytometry using fluorescent secondary IgG antibodies. did The antibodies used for comparison of relative expression patterns based on secondary IgG antibodies are as follows: CD29-PE, CD34-FITC, CD38-FITC, CD44-FITC, CD45, CD90-PE, CD105-FITC, MHC class II -FITC and IgG-FITC. About 5-8x10 5 cells per sample were used to analyze one antibody, and flow cytometry was performed using Cellquest software (FACScan, BD Biosciences, USA).
그 결과, 도 10에서 확인할 수 있듯이, 중간엽 줄기세포의 양성 발현 마커인 CD29, CD44, CD90, CD105에는 양성의 면역학적 표현형을 나타내었으며, 면역 세포 특이적 마커인 CD34, CD38, CD45, 면역 관련 마커인 MHC class II에는 음성의 면역학적 표현형을 나타내었다. 이는, 본 발명의 양막 유래 줄기세포는 중간엽 줄기세포의 특성을 가지고 있음을 시사한다.As a result, as can be seen in FIG. 10, positive immunological phenotypes were shown in CD29, CD44, CD90, and CD105, which are mesenchymal stem cell-specific markers, and immune cell-specific markers CD34, CD38, CD45, and immune-related markers. The marker, MHC class II, showed a negative immunological phenotype. This suggests that the amniotic membrane-derived stem cells of the present invention have the characteristics of mesenchymal stem cells.
실시예 5. 바이오패치의 제작Example 5. Fabrication of biopatch
1) 프라이머 처리1) Primer treatment
생분해성 PLLA(poly(L-Lactic acid)) 필름을 15 x 15 (cm)로 준비하였다. 먼저, 후술할 점착제 코팅 과정에서 세포 배양면을 보호하기 위하여 무진지(clean paper)를 이용하여 한쪽 면을 밀봉하였다. 그 다음, 점착제와 PLLA 필름 간의 친화성을 위하여 PLLA 필름 세포 배양면의 반대면에 프라이머를 처리하였다. 구체적으로, DY 39-067 Rubber Additive(SILASTIC쪠)를 솜에 적셔 PLLA 필름에 고르게 1회 처리한 뒤, 40 ℃ 오븐에 넣어 5분간 건조시켜 충분히 용매를 날려주었다.A biodegradable poly(L-Lactic acid) (PLLA) film was prepared with a size of 15 x 15 (cm). First, one side was sealed using clean paper to protect the cell culture side during the adhesive coating process to be described later. Then, for affinity between the adhesive and the PLLA film, a primer was treated on the opposite side of the cell culture side of the PLLA film. Specifically, after soaking cotton with DY 39-067 Rubber Additive (SILASTIC) and evenly treating it once on the PLLA film, it was dried in an oven at 40 ° C for 5 minutes, and the solvent was blown off sufficiently.
2) 점착제 코팅2) Adhesive coating
이액형 실리콘 점착제인 7-9700 Soft Skin Adhesive(DOW CORNING®) A와 B형을 45:55 비율로 배합하고, 교반기(overhead stirrer)를 이용하여 1000 rpm으로 2분간 교반하였다. 그 다음, 충분히 기포가 가라앉는 것을 확인한 뒤 자동도공기(Ocean science) 및 베이커 어플리케이터(baker applicator)를 이용하여 코팅하였다. 코팅은 15 x 15 (cm) 필름 기준 점착제 3 g을 필름에 올린 후 베이커 어플리케이터를 50 μm로 설정하고 50 mm/sec 속도로 수행하였다. 점착제가 코팅된 필름은 40 ℃ 오븐에 넣어 6시간 동안 경화시켰다.Two-component silicone adhesive, 7-9700 Soft Skin Adhesive (DOW CORNING ® ) A and B types were mixed in a ratio of 45:55 and stirred for 2 minutes at 1000 rpm using an overhead stirrer. Then, after confirming that bubbles had sufficiently settled, coating was performed using an automatic coating machine (Ocean science) and a baker applicator. Coating was performed at a speed of 50 mm/sec with a baker applicator set to 50 µm after placing 3 g of adhesive based on a 15 x 15 (cm) film on the film. The adhesive-coated film was cured in an oven at 40 °C for 6 hours.
3) 이형 필름 부착3) Attach release film
불소 처리된 PET(polyethylene terephthalate) 이형 필름(FL-25BMM, 기승산업)을 반자동형 망글(Ocean science)을 이용하여 부착하였다. 또한, 기포가 생기지 않도록 하기 위하여 페이더 망글의 롤 사이에 점착제가 코팅된 PLLA 필름과 불소 처리된 PET 이형 필름을 넣은 후 핸들을 조절하여 높이를 조절하였다. 롤 속도는 10 rpm으로 진행하였다.A fluorine-treated PET (polyethylene terephthalate) release film (FL-25BMM, Giseung Industry) was attached using a semi-automatic mangle (Ocean science). In addition, in order to prevent air bubbles from occurring, the adhesive-coated PLLA film and the fluorine-treated PET release film were placed between the rolls of the fader mangle, and the height was adjusted by adjusting the handle. The roll speed was 10 rpm.
4) 다공성 필름 제작4) Production of porous film
정밀 타공기를 자체 제작하여, PET 이형필름이 부착된 PLLA 필름의 휨 현상을 최소화하는 방식의 다공성 필름을 제작하였다. 다공성 필름의 한 구멍의 직경은 약 1.5마이크론으로 진행하였고, 다공성 필름의 구멍이 난 총 면적은 필름 전체 면적의 20~50 %를 차지하였다(도 11 참조). 후 공정(타공 후 공정을 의미)에도 유리한 방식의 세포 배양면 디자인을 통하여 투명한 폴리머 재질의 생분해성 필름에 세포 배양이 가능하도록 설계하였다.A precision perforation machine was manufactured in-house to produce a porous film in a way to minimize warpage of the PLLA film attached with the PET release film. The diameter of one hole in the porous film was about 1.5 microns, and the total area of the hole in the porous film accounted for 20 to 50% of the total area of the film (see FIG. 11). Cell culture was designed to enable cell culture on a biodegradable film made of transparent polymer material through the design of the cell culture surface in a way that is advantageous to the post-process (meaning the process after perforation).
5) 세포 배양면 처리5) Cell culture surface treatment
줄기세포의 배양에 적합한 세포 부착력을 유도하기 위해 초음파 처리와 플라즈마 처리를 진행하였다. 초음파 세척기로 다공성 필름을 세척하여 상온에 건조한 후, 대기압 플라즈마 처리를 진행하여 줄기세포 배양용 다공성 필름을 제작하였다. In order to induce cell adhesion suitable for stem cell culture, ultrasonic treatment and plasma treatment were performed. After washing the porous film with an ultrasonic cleaner and drying it at room temperature, atmospheric pressure plasma treatment was performed to prepare a porous film for culturing stem cells.
구체적으로, 플라즈마 처리 조건별 PLLA/SSA(Soft skin adhesive) 필름의 세포 생착률 테스트를 진행하였다. 인간 혈관내피 유래 암세포(Human hepatic adenocarcinoma cell line, sk-hep1 cells)(ATCC) 및 인간 피부 유래 섬유아세포(Human skin Fibroblast, CCD-986sk)(ATCC)의 여러 플라즈마 조건 별 세포 생착 및 생존 능력을 확인하였다.Specifically, the cell engraftment rate test of the PLLA/SSA (Soft skin adhesive) film was conducted for each plasma treatment condition. Confirmation of cell engraftment and viability of human hepatic adenocarcinoma cell line (sk-hep1 cells) (ATCC) and human skin fibroblast (CCD-986sk) (ATCC) under various plasma conditions did
이때, Plate A는 일반 세포 배양용기(cell culture plate)에 필름을 덧대지 않은 채로 세포를 넣지 않거나(-) 세포를 넣어(+) 각각 음성, 양성 대조군으로 사용하였다. 또한, Plate B는 Plate A의 재질과 같은 폴리스타이렌(polystyrene) 필름으로 또 다른 대조군으로 사용하였다. Plate C-F는 각기 다른 방식의 플라즈마 처리가 된 세포 배양 복합체(PLLA/SSA)이다. 구체적인 아르곤(lpm)/산소(sccm) 가스 조건은 하기 표 2에 나타내었다.At this time, Plate A was used as a negative and positive control, respectively, without cells (-) or with cells (+) without a film attached to a general cell culture plate. In addition, Plate B was used as another control with the same polystyrene film as Plate A. Plate C-F is a cell culture complex (PLLA/SSA) treated with different types of plasma. Specific argon (lpm) / oxygen (sccm) gas conditions are shown in Table 2 below.
도 12a 및 12b에서 확인할 수 있듯이, 모든 실험군이 대조군(+) 대비 80% 이상의 생착율을 나타내었다. 특히, Plate D에서 세포 생착률 결과가 가장 좋은 것을 확인함으로써, Plate D의 플라즈마 처리 조건으로 최종 줄기세포 배양용 다공성 필름을 제작하였다.6) 하이드로젤 제작젤라틴을 3차 증류수에 용해시킨 후, 상온에서 EDC(1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide)/NHS(N-hydroxysucciide) 가교제와 tyramine hydrochloride를 넣고 12시간 동안 교반시켜 젤라틴을 개질하였다. 용액 내의 불순물과 미반응물을 제거하기 위해 0.1 M NaCl 용액, 25% 에탄올, 3차 증류수를 사용하여 투석을 진행하고, 투석이 끝난 용액은 동결건조하여 티라민으로 개질된 젤라틴 스펀지를 제조하였다. 티라민으로 개질된 젤라틴을 3차 증류수에 6wt%, 35 ℃로 녹였다. 별도로, 10 ml 세포배양액을 동결건조한 파우더를 4 ℃에서 3차 증류수 1 ml에 녹였다. 각 용액을 1:1로 상온에서 섞은 후 HRP(horseradish peroxidase)와 과산화수소(hydrogen peroxide, H2O2)를 최종농도가 각각 0.55 units/ml, 0.025 ul/ml가 되도록 넣어 섞은 뒤 실리콘 몰드에 부어 경화시켰다(도 13 참조).As can be seen in Figures 12a and 12b, all experimental groups showed an engraftment rate of 80% or more compared to the control group (+). In particular, by confirming that the cell engraftment rate was the best in Plate D, a porous film for final stem cell culture was produced under the plasma treatment conditions of Plate D. 6) Hydrogel production After dissolving gelatin in tertiary distilled water, add EDC (1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide)/NHS (N-hydroxysucciide) crosslinking agent and tyramine hydrochloride at room temperature and stir for 12 hours. to modify gelatin. In order to remove impurities and unreacted substances in the solution, dialysis was performed using 0.1 M NaCl solution, 25% ethanol, and tertiary distilled water, and the dialysis solution was lyophilized to prepare a gelatin sponge modified with tyramine. Gelatin modified with tyramine was dissolved in tertiary distilled water at 6wt% and 35 °C. Separately, lyophilized powder of 10 ml cell culture medium was dissolved in 1 ml of tertiary distilled water at 4 °C. After mixing each solution 1:1 at room temperature, add HRP (horseradish peroxidase) and hydrogen peroxide (H 2 O 2 ) to a final concentration of 0.55 units/ml and 0.025 ul/ml, respectively, mix and pour into a silicone mold. Cured (see FIG. 13).
7) 바이오패치 제작7) Production of biopatch
상기에서 제작된 줄기세포 배양용 다공성 필름(도 14의 A), 세포배양액이 함유된 하이드로젤(도 14의 B) 및 피부 부착용 점착제(도 14의 C)를 순서대로 배치시켜 바이오패치를 제작하였다.A biopatch was fabricated by sequentially arranging the porous film for stem cell culture prepared above (FIG. 14A), the hydrogel containing the cell culture medium (FIG. 14B), and the skin adhesive (FIG. 14C). .
실시예 6. 태반 유래 줄기세포의 바이오패치에의 적용Example 6. Application of placenta-derived stem cells to biopatch
1) 세포 준비1) Cell preparation
3~5 계대 사이의 양막 유래 세포를 15 % FBS, 1 % Glutamax 및 1 % 페니실린/스트렙토마이신이 포함된 DMEM 배양 배지를 사용하여 배양하였다. 배양 접시 내의 배지를 제거하고 PBS로 세척 후 0.05% Typsin-EDTA를 이용하여 세포를 기저면에서 분리시켰다. 분리된 세포를 1x104 cells/cm2 수로 배양하였다.Amniotic membrane-derived cells between
2) 세포 형태2) cell morphology
바이오패치가 없는 배양 접시와 상기 실시예 5에서 제작된 바이오패치가 있는 배양 접시 각각에서 2일 동안 배양한 세포를 PBS로 세척 후 메탄올로 세포를 고정시켰다. 고정된 세포는 크산텐(Xanten) 및 메틸렌 블루(methylene blue)를 이용하여 각각 세포질과 세포 핵을 염색한 후, 광학현미경을 이용하여 그 형태를 관찰하였다.Cells cultured for 2 days in each of the culture dish without the biopatch and the culture dish with the biopatch prepared in Example 5 were washed with PBS and then the cells were fixed with methanol. After staining the cytoplasm and cell nuclei of the fixed cells using xanten and methylene blue, respectively, the morphology was observed using an optical microscope.
그 결과, 도 15에서 확인할 수 있듯이, 바이오패치 유무에 따른 세포의 형태에 차이가 나타나지 않았다. 이는, 바이오패치 환경에서도 세포 형태의 변화 없이 배양이 가능한 것을 시사한다.As a result, as can be seen in FIG. 15, there was no difference in cell morphology according to the presence or absence of the biopatch. This suggests that it is possible to culture without changing cell morphology even in a biopatch environment.
3) 세포 수 및 세포 생존율3) Cell number and cell viability
바이오패치가 없는 배양 접시와 상기 실시예 5에서 제작된 바이오패치가 있는 배양 접시 각각에서 48 시간 동안 배양한 줄기세포를 PBS로 세척 후 0.05% Typsin-EDTA를 이용하여 세포를 기저면에서 분리시킨 뒤 원심분리기를 이용하여 세포를 모아주었다. 모인 세포를 트리판 블루로 염색한 뒤 혈구계(hemocytometer) 위에 염색된 세포를 도포하고 광학현미경으로 관찰하였다. 염색되지 않은 세포와 염색된 세포의 수를 측정하여 그 수와 생존율을 측정하였다.Stem cells cultured for 48 hours in a culture dish without a biopatch and a culture dish with a biopatch prepared in Example 5 were washed with PBS, separated from the basal surface using 0.05% Typsin-EDTA, and centrifuged. Cells were collected using a separator. After staining the collected cells with trypan blue, the stained cells were coated on a hemocytometer and observed under an optical microscope. The number and viability were determined by counting the number of unstained and stained cells.
그 결과, 도 16에서 확인할 수 있듯이, 바이오패치 유무에 따른 세포의 증식 능력에 차이가 나타나지 않았다. 이는, 바이오패치 환경에서도 세포 증식 능력의 차이 없이 배양이 가능한 것을 시사한다.As a result, as can be seen in FIG. 16, there was no difference in the proliferative capacity of the cells according to the presence or absence of the biopatch. This suggests that culture is possible even in a biopatch environment without a difference in cell proliferation ability.
4) 세포 독성 및 세포 사멸(MTT assay, Annexin V staining)4) Cytotoxicity and cell death (MTT assay, Annexin V staining)
MTT assayMTT assay
바이오패치가 없는 배양 접시와 상기 실시예 5에서 제작된 바이오패치가 있는 배양 접시 각각에서 2일 동안 배양하고, Vybrant MTT Cell Proliferation Assay Kit(Thermo Fisher Scientific)를 이용하여 죽은 세포의 양을 비교하였다.It was cultured for 2 days in a culture dish without a biopatch and a culture dish with a biopatch prepared in Example 5, respectively, and the amount of dead cells was compared using the Vybrant MTT Cell Proliferation Assay Kit (Thermo Fisher Scientific).
Annexin V stainingAnnexin V staining
바이오패치가 없는 배양 접시와 상기 실시예 5에서 제작된 바이오패치가 있는 배양 접시 각각에서 2일 동안 배양한 세포를 PBS로 세척 후 0.05% Trpsin-EDTA를 이용하여 세포를 기저면에서 분리시킨 뒤 원심분리기를 이용하여 세포를 모아주었다. 키트(No. V13241, Invitrogen)를 이용하여 염색하고 유세포분석기를 이용하여 살아있는 세포, 세포자살(apoptosis) 및 세포괴사(necrosis)를 분석하였다.After washing the cells cultured for 2 days in a culture dish without a biopatch and a culture dish with a biopatch prepared in Example 5 with PBS, the cells were separated from the basal surface using 0.05% Trpsin-EDTA and centrifuged. The cells were collected using The cells were stained using a kit (No. V13241, Invitrogen) and analyzed for viable cells, apoptosis and necrosis using a flow cytometer.
그 결과, 도 17에서 확인할 수 있듯이, 바이오패치 유무에 따른 세포의 활성에 차이가 나타나지 않았다. 이는, 바이오패치 환경에서도 세포 증식 및 생존의 차이 없이 배양이 가능한 것을 시사한다.As a result, as can be seen in FIG. 17, there was no difference in cell activity according to the presence or absence of the biopatch. This suggests that culture can be performed without differences in cell proliferation and survival even in a biopatch environment.
실시예 7. 바이오패치형 줄기세포 치료제의 피부 치료 효능 유효성 평가Example 7. Efficacy evaluation of skin treatment efficacy of biopatch-type stem cell treatment
1) 피부 손상 동물 모델 1) Skin damage animal model
6주령의 수컷 ICR mice(두열 바이오텍, KOREA)에 대하여, 충남대학교 동물실험윤리위원회 설치·운영 규정에 의거하여(승인번호, 202103A-CNU-072) 유효성 평가를 진행하였다. 마우스는 5주령에 반입 후 일주일간 안정화시킨 후, 마취제(80 mg/kg ketamine + 12 mg/kg xylazine)를 복강 내 투여하여 마취하고 Biopsy punch를 통해 창상을 유도하였다.For 6-week-old male ICR mice (Doo-yeol Biotech, KOREA), efficacy evaluation was conducted in accordance with the establishment and operation regulations of the Animal Experimentation Ethics Committee of Chungnam National University (approval number, 202103A-CNU-072). The mice were brought in at 5 weeks of age, stabilized for one week, anesthetized by intraperitoneal administration of an anesthetic (80 mg/kg ketamine + 12 mg/kg xylazine), and wounds were induced through a biopsy punch.
2) 바이오패치형 줄기세포 치료제 이식2) Implantation of biopatch-type stem cell treatment
상기 실시예 5에서 제작된 바이오패치에 상기 실시예 6-1)의 양막 유래 줄기세포를 배양한 바이오패치형 줄기세포 치료제를 피부 손상(창상) 부위에 부착 후 멸균 방수 필름(TegadermTM)으로 덮은 뒤, 피부가 수축되지 않도록 직경 6 mm의 구멍을 낸 둥근 모양의 실리콘을 피부에 부착시킨 후 봉합사로 꿰매어 고정시켜 유효성 평가를 진행하였다(도 18 참조). 줄기세포가 배양되지 않은 바이오패치를 대조군(Control)로 설정하였다.After attaching the biopatch-type stem cell therapeutic agent in which the amniotic membrane-derived stem cells of Example 6-1) were cultured to the biopatch prepared in Example 5 was attached to the skin damaged (wound) area, it was covered with a sterile waterproof film (Tegaderm TM ). , To prevent contraction of the skin, round-shaped silicone with a diameter of 6 mm was attached to the skin, and then it was sewn and fixed with a suture to evaluate effectiveness (see FIG. 18). A biopatch in which stem cells were not cultured was set as a control.
3) 결과3) Results
피부 손상 면적 변화 확인Check the change in skin damage area
상기 바이오패치형 줄기세포 치료제가 이식된 피부 손상 부위에 대하여, 치료제 이식 후 0, 3, 7, 10 또는 14일에 상처 면적을 측정하였으며(도 19a), Image J 프로그램을 이용하여 상처 면적 변화를 확인하였다(도 19b).Regarding the damaged skin area to which the biopatch-type stem cell therapeutic agent was transplanted, the wound area was measured at 0, 3, 7, 10, or 14 days after transplantation of the therapeutic agent (FIG. 19a), and the change in wound area was confirmed using the Image J program. (Fig. 19b).
그 결과, 도 19a 및 19b에서 확인할 수 있듯이, 바이오패치형 줄기세포 치료제 처리 군에서 수술 후 7, 10, 14일에 통계적으로 피부 손상 부위 면적이 유의하게 감소됨을 알 수 있었다.As a result, as can be seen in FIGS. 19a and 19b, it was found that the area of the skin damaged area was statistically significantly reduced at 7, 10, and 14 days after surgery in the biopatch-type stem cell treatment group.
피부 재생 효과 확인Check skin regeneration effect
상기 바이오패치형 줄기세포 치료제가 이식된 피부 손상 부위에 대하여, 치료제 이식 후 3 또는 7일에 상처 부분의 피부조직을 채취하였다. 채취한 조직을 10% 포르말린(formalin) 용액으로 고정 후 파라핀(paraffin) 블록을 제작하였다. 제작된 파라핀 블록에서 4 um 두께로 절단한 슬라이드를 Hematoxylin and eosin(H&E) 염색하여 과립조직형성(granulation tissue formation, 도 20a) 및 피부의 재상피화(re-epithelialization, 도 20b)를 평가하였다. 피부의 재상피화는 정상 피부 조직 끝부분(Skin margin)에서부터 재상피화가 진행된 부분까지의 길이를 Image J 프로그램을 이용하여 수치화하였다.Regarding the damaged skin area to which the biopatch-type stem cell therapeutic agent was implanted, skin tissue of the wound portion was collected 3 or 7 days after transplantation of the therapeutic agent. After fixing the collected tissue with a 10% formalin solution, a paraffin block was prepared. Hematoxylin and eosin (H&E) staining of slides cut to a thickness of 4 μm from the prepared paraffin block was performed to evaluate granulation tissue formation (Fig. 20a) and skin re-epithelialization (Fig. 20b). The skin re-epithelialization was quantified by using the Image J program as the length from the skin margin to the re-epithelialized area.
그 결과, 도 20a 및 20b에서 확인할 수 있듯이, 피부 손상 3일 후 대조군은 0.27±0.05 mm, 치료제 이식 그룹에서는 0.51±0.04 mm의 재상피화가 일어났으며, 피부 손상 7일 후 대조군에서는 0.55±0.05 mm, 치료제 이식 그룹에서는 1.07±0.12 mm의 재상피화가 일어났다. 결과적으로, 본 발명의 바이오패치형 줄기세포 치료제 처리 군에서 상처 조직의 재상피화가 통계적으로 유의하게 촉진됨을 알 수 있었다.As a result, as can be seen in FIGS. 20a and 20b, after 3 days of skin damage, re-epithelialization of 0.27±0.05 mm in the control group and 0.51±0.04 mm in the treatment implantation group occurred, and 0.55±0.05 mm in the control group after 7 days of skin damage. mm, 1.07±0.12 mm of re-epithelialization occurred in the treatment implantation group. As a result, it was found that re-epithelialization of scar tissue was statistically significantly promoted in the group treated with the biopatch-type stem cell therapy of the present invention.
본 발명은 태반-유래 줄기세포를 함유하는 피부 재생용 바이오패치형 세포치료제에 관한 것이다.The present invention relates to a biopatch-type cell therapy product for skin regeneration containing placenta-derived stem cells.
<110> mkbiotech.co.ltd<110> mkbiotech.co.ltd
The Industry & Academic Cooperation in Chungnam National University (IAC) The Industry & Academic Cooperation in Chungnam National University (IAC)
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<213> Artificial Sequence<213> artificial sequence
<220><220>
<223> Klf4 primer-F<223> Klf4 primer-F
<400> 7<400> 7
acctcgcctt acacatgaag 20
<210> 8<210> 8
<211> 21<211> 21
<212> DNA<212> DNA
<213> Artificial Sequence<213> artificial sequence
<220><220>
<223> Klf4 primer-R<223> Klf4 primer-R
<400> 8<400> 8
tggtttcctc attgtctcct g 21tggtttcctc attgtctcct g 21
<210> 9<210> 9
<211> 21<211> 21
<212> DNA<212> DNA
<213> Artificial Sequence<213> artificial sequence
<220><220>
<223> PAX6 primer-F<223> PAX6 primer-F
<400> 9<400> 9
tgtttgcccg agaaagacta g 21tgtttgcccg agaaagacta g 21
<210> 10<210> 10
<211> 21<211> 21
<212> DNA<212> DNA
<213> Artificial Sequence<213> artificial sequence
<220><220>
<223> PAX6 primer-R<223> PAX6 primer-R
<400> 10<400> 10
agaggtgaag gatgaaacag g 21agaggtgaag gatgaaacag g 21
<210> 11<210> 11
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> artificial sequence
<220><220>
<223> ALCAM primer-F<223> ALCAM primer-F
<400> 11<400> 11
agaaggcagg aagtttgtcg 20
<210> 12<210> 12
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> artificial sequence
<220><220>
<223> ALCAM primer-R<223> ALCAM primer-R
<400> 12<400> 12
ggaaagttga ggattgtgcg 20
<210> 13<210> 13
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> artificial sequence
<220><220>
<223> Integrin-b1 primer-F<223> Integrin-b1 primer-F
**
**
*<400> 13*<400> 13
cttattggcc ttgcattgct 20
<210> 14<210> 14
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> artificial sequence
<220><220>
<223> Integrin-b1 primer-R<223> Integrin-b1 primer-R
<400> 14<400> 14
ttccctcgta cttcggattg 20
<210> 15<210> 15
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> artificial sequence
<220><220>
<223> HCAM primer-F<223> HCAM primer-F
<400> 15<400> 15
atcctcacgt ccaacacctc 20atcctcacgt ccaaccctc 20
<210> 16<210> 16
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> artificial sequence
<220><220>
<223> HCAM primer-R<223> HCAM primer-R
<400> 16<400> 16
ctcgcctttc ttggtgtagc 20ctcgcctttc ttggtgtagc 20
<210> 17<210> 17
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> artificial sequence
<220><220>
<223> Endoglin primer-F<223> Endoglin primer-F
<400> 17<400> 17
aagagctcat ctcgagtctg 20
<210> 18<210> 18
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> artificial sequence
<220><220>
<223> Endoglin primer-R<223> Endoglin primer-R
**
<400> 18<400> 18
tgacgaccac ctcattactg 20
Claims (14)
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KR1020210176353A KR102439335B1 (en) | 2021-12-10 | 2021-12-10 | Bio-patch type cell therapy product for Regenerating skin comprising Placenta-derived stem cells |
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KR100818214B1 (en) * | 2006-09-29 | 2008-04-01 | 재단법인서울대학교산학협력재단 | Amniotic membrane or decidual-derived multipotent stem cells of human placental tissue and a method of manufacturing the same |
KR20130053609A (en) * | 2011-11-15 | 2013-05-24 | 한양대학교 산학협력단 | Method for extracting extracelluar matrix from placenta and scaffold for skin wound treatment using material extracted by the same |
KR101523040B1 (en) * | 2012-06-26 | 2015-05-26 | 라정찬 | Method for Preparing High Concentration of Stem Cells |
JP2018512959A (en) * | 2015-04-23 | 2018-05-24 | ユニバーシティ オブ フロリダ リサーチ ファウンデーション,インコーポレイテッド | Bilayer device for improved healing |
JP2021035976A (en) * | 2014-10-02 | 2021-03-04 | ウェイク フォレスト ユニバーシティー ヘルス サイエンシズ | Amniotic membrane powder and its use in wound healing and tissue engineering structures |
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US20090004271A1 (en) * | 2007-06-29 | 2009-01-01 | Brown Laura J | Morselized foam for wound treatment |
KR101843293B1 (en) | 2016-12-07 | 2018-03-29 | 서울대학교산학협력단 | Biopatch |
JP6933394B2 (en) * | 2018-09-06 | 2021-09-08 | マリン エッセンス バイオサイエンシズ コーポレーション オブ ユーエスエーMarine Essence Biosciences Corporation of USA | Biomaterial devices and topical compositions for inducing tissue regeneration |
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KR100818214B1 (en) * | 2006-09-29 | 2008-04-01 | 재단법인서울대학교산학협력재단 | Amniotic membrane or decidual-derived multipotent stem cells of human placental tissue and a method of manufacturing the same |
KR20130053609A (en) * | 2011-11-15 | 2013-05-24 | 한양대학교 산학협력단 | Method for extracting extracelluar matrix from placenta and scaffold for skin wound treatment using material extracted by the same |
KR101523040B1 (en) * | 2012-06-26 | 2015-05-26 | 라정찬 | Method for Preparing High Concentration of Stem Cells |
JP2021035976A (en) * | 2014-10-02 | 2021-03-04 | ウェイク フォレスト ユニバーシティー ヘルス サイエンシズ | Amniotic membrane powder and its use in wound healing and tissue engineering structures |
JP2018512959A (en) * | 2015-04-23 | 2018-05-24 | ユニバーシティ オブ フロリダ リサーチ ファウンデーション,インコーポレイテッド | Bilayer device for improved healing |
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